Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Oxidative stress facilitates exogenous mitochondria internalization and survival in retinal ganglion precursor-like cells.

Ocular cells are highly dependent on mitochondrial function due to their high demand of energy supply and their constant exposure to oxidative stress. Indeed, mitochondrial dysfunction is highly implicated in various acute, chronic, and genetic disorders of the visual system. It has recently been shown that mitochondrial transplantation (MitoPlant) temporarily protects retinal ganglion cells (RGCs) from cell death during ocular ischemia. Here, we characterized MitoPlant dynamics in retinal ganglion precursor-like cells, in steady state and under oxidative stress. We developed a new method for detection of transplanted mitochondria using qPCR, based on a difference in the mtDNA sequence of C57BL/6 and BALB/c mouse strains. Using this approach, we show internalization of exogenous mitochondria already three hours after transplantation, and a decline in mitochondrial content after twenty four hours. Interestingly, exposure of target cells to moderate oxidative stress prior to MitoPlant dramatically enhanced mitochondrial uptake and extended the survival of mitochondria in recipient cells by more than three fold. Understanding the factors that regulate the exogenous mitochondrial uptake and their survival may promote the application of MitoPlant for treatment of chronic and genetic mitochondrial diseases.

In-vivo imaging for assessing tumor growth in mouse models of ocular melanoma.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Cellular metabolism constrains innate immune responses in early human ontogeny.

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic ß-Cells.

In pancreatic ß-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal ß-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress ß-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of ß-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca(2+) responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional ß-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca(2+) influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of ß-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in ß-cells. These noncanonical roles of Bcl-2 may be important for ß-cell function and survival under conditions of high metabolic demand.

Nitric oxide, can it be only good? Increasing the antioxidant properties of nitric oxide in hepatocytes by YC-1 compound.

The aim of the study was to evaluate the effect of Nitric oxide (NO) on redox changes and fat accumulation in hepatocytes. AML-12 hepatocytes were exposed to the NO donor Diethylenetriamine-NONOate (DETA-NO). DETA-NO led to a dose- and time-dependent increase in lipid accumulation in the cells, measured by Nile red fluorescence. Exposure of the cells to 1mM DETA-NO for 24h increased reactive oxygen species production, mainly peroxides. At the same time, NO induced elevation of reduced glutathione (GSH) and a mild activation of the antioxidant transcription factors Hypoxia-inducible factor 1α (HIF1α) and NF-E2 related factor 2 (Nrf-2). We used 100 µM YC-1 to inhibit HIF1α activity and induce activation of soluble Guanylate Cyclase (sGC). YC-1 alone did not affect fat accumulation, and only moderately increased the expression of Nrf-2-targeted genes Heme oxygenase 1 (Hmox1), NAD(P)H dehydrogenase (quinone 1) (Nqo1) and Glutathione S-transferase α1 (Gstα1). However, YC-1 abolished the negative effect of NO on fat accumulation when administered together. Strikingly, YC-1 potentiated the effect of NO on Nrf-2 activation, thus increasing dramatically the antioxidant properties of NO. Moreover, YC-1 intensified the effect of NO on the expression of peroxisome-proliferator-activated receptor-gamma co-activator 1α (PGC1α) and mitochondrial biogenesis markers. This study suggests that YC-1 may shift the deleterious effects of NO into the beneficial ones, and may improve the antioxidant properties of NO.

Fatty liver is associated with impaired activity of PPARγ-coactivator 1α (PGC1α) and mitochondrial biogenesis in mice.

Accumulating evidence indicates that mitochondria have a key role in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Histological studies demonstrated accumulation of fat vacuoles in up to 90% of hepatocytes in mice fed the CDE diet for 14 days. In addition, a decrease in mitochondrial levels, together with an increase in superoxide radicals' levels were observed, indicating elevation of oxidative stress in hepatocytes. ATP levels were decreased in livers from CDE-fed mice after overnight fasting. This was accompanied by a compensative and significant increase in peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) mRNA levels in comparison to control livers. However, there was a reduction in PGC1α protein levels in CDE-treated mice. Moreover, the expression of mitochondrial biogenesis genes nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M) and mitochondrial transcription factor B2 (TFB2M), which are all regulated by PGC1α activity, remained unchanged in fasted CDE-treated mice. These results indicate impaired activity of PGC1α. The impaired activity was further confirmed by chromatin immunoprecipitation analysis, which demonstrated decreased interaction of PGC1α with promoters containing NRF-1 and NRF-2 response elements in mice fed the CDE diet. A decrease in PGC1α ability to activate the expression of the gluconeogenic gene phosphoenol-pyruvate carboxykinase was also observed. This study demonstrates, for the first time, that attenuated mitochondrial biogenesis in steatotic livers is associated with impaired biological activity of PGC1α.

Impaired liver glucose production in a murine model of steatosis and endotoxemia: protection by inducible nitric oxide synthase.

This study hypothesized that upregulation of inducible nitric oxide synthase (iNOS) would preserve the metabolic status of the liver under conditions of steatosis and acute inflammation. Wild-type C57BL/6J and C57BL/6 iNOS-knockout (-/-) mice were fed a choline-deficient ethionine-supplemented diet (CDE). Mice were also injected with 5 mg/kg lipopolysaccharide (LPS) to induce endotoxemia. Consumption of the CDE diet led to steatosis of the liver and decreased expression of the gluconeogenic genes compared with controls. LPS treatment exacerbated these effects because of inhibition of PGC-1alpha expression, which resulted in hypoglycemia. In steatotic livers, LPS-induced iNOS expression was enhanced. Comparison between wild-type and iNOS-knockout mice under these conditions demonstrated a protective role of iNOS against fatal hypoglycemia. Nitric oxide (NO) signaling effects were confirmed by treatment of hepatocytes in culture with an NO donor, which resulted in increased expression of PGC-1alpha and gluconeogenic genes. In conclusion, iNOS was found to act as a protective protein and provides a possible mechanism by which the liver preserves glucose homeostasis under stress.

Triacylglycerol-induced impairment in mitochondrial biogenesis and function in J774.2 and mouse peritoneal macrophage foam cells.

The aim of this study was to detect mitochondrial alterations in J774.2 macrophages and mouse peritoneal macrophages (MPM) foam cells. J774.2 and MPM cells were exposed to triacylglycerol (TG) emulsion (1 mg/ml) for induction of fat accumulation. Impairment of mitochondrial function was reflected by reduced cellular ATP production and decreased expression of subunits of mitochondrial complexes I and III. The expression of subunit IV of complex IV remained unchanged, however, the content of its precursor in cells increased. Inhibitors of mitochondrial complexes, rotenone (0.1 microM) and myxothiazol (25 nM), protected the viability in TG-loaded macrophages. The exposure to TG caused downregulation of PPARgamma coactivator (PGC)-1alpha and nuclear respiratory factor (NRF)-1. Activation of peroxisome proliferator-activated receptors attenuated reactive oxygen species production in the foam cells. Treatment with antioxidant N-acetylcysteine (NAC) prevented lipid-mediated mitochondrial and cellular damage. In conclusion, this study demonstrates the important role of mitochondrial biogenesis dysfunction in TG-induced lipotoxicity in macrophages.

Selenium supplementation increases liver MnSOD expression: molecular mechanism for hepato-protection.

Selenium is recognized as essential in animal and human nutrition. Several hypotheses have been advanced for its biological activity. The aim of this study was to investigate the in vivo effect of selenium on rat liver manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, under naïve and inflammatory conditions. Rats received sodium selenite supplementation and LPS injection. Whole-liver samples, isolated hepatocytes, Kupffer cells and blood samples were subjected to protein, RNA and biochemical analysis. Liver enrichment with selenium increased whole-liver MnSOD levels due to an increase in MnSOD transcription in hepatocytes. This was due to an increase in the ratio of specificity protein 1 to activating enhancer binding protein 2 DNA-binding activity. The inflammatory stimulus further elevated MnSOD levels in the whole-liver that was abrogated in sodium selenite supplementation due to reduced transcription of MnSOD in Kupffer cells. Moreover, selenium enrichment decreased Kupffer cells IL-6 transcription in LPS-injected animals. Anti-inflammatory activity of selenium was demonstrated by normalized blood levels of ALT and IL-6 in LPS-injected animals. In conclusion, selenium up-regulates hepatocytes MnSOD expression, probably improving their anti-oxidant defense, while decreasing MnSOD and IL-6 transcription in Kupffer cells in the presence of inflammatory stimuli, attenuating their inflammatory response. This selective mechanism may explain the anti-inflammatory and hepato-protective effect of selenium.

ROS-production-mediated activation of AP-1 but not NFkappaB inhibits glutamate-induced HT4 neuronal cell death.

Aside from their deleterious effect, reactive oxygen species (ROS) can function as small messenger molecules during physiologic processes. ROS have been shown to activate the transcription nuclear factor kappa B (NFkappaB) and activator protein 1 (AP-1). Exposure of HT4 neuronal cells to 10 mM glutamate results in cell death after 12 h. Here we show that glutamate treatment leads to an increase in ROS production and activation of AP-1, but not NFkappaB. 12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C and an inducer of NFkappaB and AP-1, protected the cells. This protective effect was preceded by increased production of ROS compared with glutamate alone, which was accompanied by a synergistic increase in AP-1, but not NFkappaB activity. We used all-trans-retinoic acid (ATRA), overexpression of retinoic acid receptor alpha (RARalpha) and a decoy oligonucleotide inclusion assay to suppress AP-1 activity. NFkappaB was inhibited by using a super suppressor (IkappaBalphaDeltaN-transfected cells). Inhibition of AP-1, but not NFkappaB resulted in increased cellular vulnerability to glutamate. Inhibition of AP-1 activity was coincident with a decrease in ROS production. Thus, although ROS are significant to the cell-death effect induced by glutamate, they also activate protective pathways mediated by increasing AP-1 activity, and not that of NFkappaB.

Selenium attenuates expression of MnSOD and uncoupling protein 2 in J774.2 macrophages: molecular mechanism for its cell-death and antiinflammatory activity.

Selenium can activate cell death. However, the mechanism of action is not yet fully defined. We hypothesized that selenium may impede mitochondrial superoxide dismutation to H2O2 and O2, leading to cell death in macrophages and that this effect may be relevant to antiinflammatory treatment by selenium. In this study, the mechanism of action of selenium was investigated in nonactivated and activated (immune-stimulated) J774.2 macrophages. Sodium selenite treatment decreased dichlorodihydrofluorescein-reacting intracellular reactive oxygen species (ROS) (mainly peroxides and hydroxyl radicals), with no correlation to glutathione peroxidase activity. However, selenite decreased the transcription and expression of manganese superoxide dismutase (MnSOD) and uncoupling protein 2 (UCP2). This cellular effect was due to inhibition of specificity protein-1 (Sp1) binding to its DNA binding site. Following immune stimulation of macrophages using lipopolysaccharides plus interferon-gamma, MnSOD was up-regulated. Activated macrophages showed higher mitochondrial membrane potential, intracellular ROS levels, and cellular resistance to cell death. Selenite treatment attenuated all of these parameters. Selenite prevented nuclear factor-kappaB (NF-kappaB) activation as a mechanism of its inhibitory activity on MnSOD expression in the immune-stimulated cells. In addition, overexpression of human MnSOD protected against death induced by selenite treatment. It is therefore concluded that selenium at high nanomolar to low micromolar concentrations shifts the balance between inflammatory response and cell death toward the latter, through a direct effect on the transcription factors Sp1 and NF-kappaB, and down-regulation of MnSOD and UCP2.