Shani Shilo

MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue

AIMS MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression. METHODS AND RESULTS Following 2 and 7 h of IR or sham operation, myocardial tissue was collected and subjected to miRNA expression profiling and quantification using a Bioarray system that screens for human-, mice-, rat-, and Ambi-miR. Data mining and differential analyses resulted in 13 miRs that were up-regulated on day 2, 9 miRs that were up-regulated on day 7, and 6 miRs that were down-regulated on day 7 post-IR. Results randomly selected from expression profiling were validated using real-time PCR. Tissue elements laser-captured from the infarct site showed marked induction of miR-21. In situ hybridization studies using locked nucleic acid miR-21-specific probe identified that IR-inducible miR-21 was specifically localized in the infarct region of the IR heart. Immunohistochemistry data show that cardiac fibroblasts (CFs) are the major cell type in the infarct zone. Studies with isolated CFs demonstrated that phosphatase and tensin homologue (PTEN) is a direct target of miR-21. Modulation of miR-21 regulated expression of matrix metalloprotease-2 (MMP-2) via a PTEN pathway. Finally, we noted a marked decrease in PTEN expression in the infarct zone. This decrease was associated with increased MMP-2 expression in the infarct area. CONCLUSION This work constitutes the first report describing changes in miR expression in response to IR in the mouse heart, showing that miR-21 regulates MMP-2 expression in CFs of the infarct zone via a PTEN pathway.

Evidence for the involvement of miRNA in redox regulated angiogenic response of human microvascular endothelial cells.

Objective—A Dicer knockdown approach was used to test the significance of miRNA in regulating the redox state and angiogenic response of human microvascular endothelial cells (HMECs). Methods and Results—Lowering of miRNA content by Dicer knockdown induced vascular endothelial growth factor expression but diminished the angiogenic response of HMECs as determined by cell migration and Matrigel tube formation. Such impairment of angiogenic response in the Matrigel was rescued by exogenous low micromolar H2O2. Dicer knockdown HMECs demonstrated lower inducible production of reactive oxygen species (ROS) when activated with either phorbol ester, tumor necrosis factor-&agr;, or vascular endothelial growth factor. Limiting the production of ROS by antioxidant treatment or NADPH oxidase knockdown approaches impaired angiogenic responses. Experiments to identify how ROS production is limited by Dicer knockdown identified lower expression of p47phox protein in these cells. This lowering of cellular miRNA content induced expression of the transcription factor HBP1, a suppressor transcription factor that negatively regulates p47phox expression. Knockdown of HBP1 restored the angiogenic response of miRNA-deficient HMECs. Conclusion—This study provides the first evidence that redox signaling in cells is subject to regulation by miRNA. Specifically, p47phox of the NADPH oxidase complex has been identified as one target that regulates the angiogenic properties of endothelial cells.

Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species

AbstractThe role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.

Selenite sensitizes mitochondrial permeability transition pore opening in vitro and in vivo: a possible mechanism for chemo-protection.

There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1-10 microM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N -acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16 p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.

Membrane depolarization of isolated rat liver mitochondria attenuates permeability transition pore opening and oxidant production.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.

Selenite Activates Caspase-Independent Necrotic Cell Death in Jurkat T Cells and J774.2 Macrophages by Affecting Mitochondrial Oxidant Generation

Sodium selenite, a common dietary form of selenium, is recognized as essential in animal and human nutrition. Mechanisms regulating the inflammatory response of the immune system involve regulation of apoptosis and control of reactive oxygen species (ROS) production. In this study, the effect of sodium selenite on ROS production and cell-death rates in macrophages and T cells was investigated. Exposing Jurkat T cells or J774.2 macrophages to >5 micro M sodium selenite induced cell death. In both Jurkat T cells and J774.2 macrophages, rapid loss of the cells capacity to generate dichlorofluorescein-sensitive ROS preceded cell death. The main cellular source of ROS was found to be the mitochondria electron-transfer chain. DEVDase activity in the cells remained unchanged and even decreased with time, as well as DNA fragmentation level, which was almost unaffected, indicating cell death with necrotic characteristics. tert-Butyl hydroperoxide at a concentration of 5 micro M was beneficial in attenuating the rate of cell death. The superoxide scavenger Tiron was tested for its ability to protect the cells against selenium. Tiron completely protected the J774.2 macrophage cell line against selenium and attenuated the cell death effect in Jurkat T cells. In the presence of the superoxide dismutase-mimicking compound tempol, seleniums macrophage-killing effect was inhibited. Therefore, our results show that, at least in vitro, selenite induces changes in the balance between mitochondrial superoxide and hydrogen peroxide production, which can facilitate cell death in immune system cells. This may be one mechanism by which selenium down-regulates the immune systems inflammatory response and protects against overproduction of peroxides.