Reactive Oxygen Species-dependent Manner Research Articles

Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death.

Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H2O2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN-mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis.

4,4'-diaponeurosporene, a C30 carotenoid, effectively activates dendritic cells via CD36 and NF-κB signaling in a ROS independent manner.

Carotenoids could be divided into C30 carotenoids and C40 carotenoids. The immune functions of C40 carotenoids had been extensively researched, while those of C30 carotenoids still remain unclear. In this study, the immune functions of a biosynthetic C30 carotenoid, 4,4′-diaponeurosporene (Dia), were identified on dendritic cells (DCs). DCs treated with 1 μM Dia for 24 h showed morphologic and phenotypic characteristics of mature state and had an increased production of IL-6, IL-10, IL-12p70 and TNFα, while β-carotene had a suppressive effect on DCs maturation. Moreover, Dia promoted antigen uptake of DCs in vitro and increased the quantity of antigen loaded DCs in mesenteric lymph nodes (MLN). Dia-treated DCs also had an enhanced ability to stimulate T cell proliferation and Th1 polarization. Further researches showed that Dia activated DCs via CD36 as well as ERK, JNK, and NF-κB signals in a reactive oxygen species (ROS) independent manner.

17β-Oestradiol enhances the expansion and activation of myeloid-derived suppressor cells via signal transducer and activator of transcription (STAT)-3 signalling in human pregnancy.

During a successful pregnancy, the maternal immune system plays a critical role in maintaining immunotolerance towards semi-allogeneic fetal antigens. Recent studies have indicated that myeloid-derived suppressor cells (MDSCs) are active players in establishing fetal-maternal tolerance; however, the underlying mechanism remains poorly understood. In this study, we observed a significant expansion of monocytic MDSCs (M-MDSCs) in the peripheral blood of pregnant women, which suppressed T cell responses in a reactive oxygen species-dependent manner and required cell-cell contact. The number of M-MDSCs correlated positively with serum oestrogen and progesterone levels. Administration of 17β-oestradiol, but not progesterone, enhanced both the expansion and suppressive activity of M-MDSCs through signal transducer and activator of transcription (STAT)-3. Pretreatment with STAT-3 inhibitor JSI-124 almost completely abrogated the effects of 17β-oestradiol on MDSCs. Collectively, these results demonstrate that 17β-oestradiol-induced STAT-3 signalling plays an important role in both the expansion and activation of MDSCs during human pregnancy, which may benefit the development of novel therapeutic strategies for prevention of immune-related miscarriage.

Social amoebae trap and kill bacteria by casting DNA nets.

Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans.

Myeloid-derived suppressor cells are essential for maintaining feto-maternal immunotolerance via STAT3 signaling in mice.

Maternal immune system tolerance to the semiallogeneic fetus is essential for a successful pregnancy; however, the mechanisms underlying this immunotolerance have not been fully elucidated. Here, we demonstrate that myeloid-derived suppressor cells play an important role in maintaining feto-maternal tolerance. A significant expansion of granulocytic myeloid-derived suppressor cells was observed in multiple immune organs and decidual tissues from pregnant mice. Pregnancy-derived granulocytic myeloid-derived suppressor cells suppressed T cell responses in a reactive oxygen species-dependent manner and required direct cell-cell contact. Mechanistic studies showed that progesterone facilitated differentiation and activation of granulocytic myeloid-derived suppressor cells, mediated through STAT3 signaling. The STAT3 inhibitor JSI-124 and a specific short hairpin RNA completely abrogated the effects of progesterone on granulocytic myeloid-derived suppressor cells. More importantly, granulocytic myeloid-derived suppressor cell depletion dramatically enhanced the abortion rate in normal pregnant mice, whereas adoptive transfer of granulocytic myeloid-derived suppressor cells clearly reduced the abortion rate in the CBA/J X DBA/2J mouse model of spontaneous abortion. These observations collectively demonstrate that granulocytic myeloid-derived suppressor cells play an essential role in the maintenance of fetal immunotolerance in mice. Furthermore, our study supports the notion that in addition to their well-recognized roles under pathologic conditions, myeloid-derived suppressor cells perform important functions under certain physiologic circumstances.

Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis

ObjectiveThe influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory...

Swift Intrahepatic Accumulation of Granulocytic Myeloid-Derived Suppressor Cells in a Humanized Mouse Model of Toxic Shock Syndrome.

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with 'systemic' immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of 'local' immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b(+)Gr-1(high)Ly-6C(+) granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species-dependent manner, and ex vivo-generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

IL-17A up-regulates expression of endothelial tissue factor in liver cirrhosis via the ROS/p38 signal pathway

Interleukin-17A (IL-17A), an inflammatory cytokine, is elevated in liver cirrhosis. Inflammation and coagulation dysfunction are closely related. Tissue factor (TF) is a bridge between endothelial activation, blood coagulation and inflammation. The aims of the present study were to evaluate endothelial TF expression in liver cirrhosis and identify the possible underlying role of IL-17A in TF expression. In the present study, we found that TF expression was increased on endothelium of splenic vein from cirrhotic patients and significantly correlated with intima/media ratios of splenic vein and coagulation parameters. Serum levels of IL-17A were significantly higher in cirrhotic patients as compared with normal controls. Cirrhotic serum and IL-17A stimulated TF expression in HUVECs, which was reduced by blockade of IL-17A, p38, and reactive oxygen species (ROS). Taken together, our data show that enhanced expression of endothelial TF, which plays an important role in coagulopathy and splenic vein remodeling in liver cirrhosis, is induced by IL-17A in a ROS dependent manner.

Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors.

Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas’s disease can limit infection by affecting the infectivity/pathogenicity of the parasite.

Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

Daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner.

This study aims to evaluate the anti-cancer effect of daucosterol and explore its possible mechanism. MTT and colony formation assay were performed to determine the effect of daucosterol on cancer cell proliferation in vitro. H22 allograft model was used for the assessment of its anti-cancer activity in vivo. Intracellular generation of reactive oxygen species (ROS) was measured using DCFH-DA probe with flow cytometry system and a laser scanning confocal microscope. LC3 (microtubule-associated protein 1 light chain 3)-II conversion was monitored with immunofluorescence and immunoblotting to demonstrate daucosterol-induced autophagy. We found that daucosterol inhibits the proliferation of human breast cancer cell line MCF-7 and gastric cancer cell lines MGC803, BGC823 and AGS in a dose-dependent manner. Furthermore, daucosterol inhibits murine hepatoma H22 cell growth in ICR mice. Daucosterol treatment induces intracellular ROS generation and autophagy, but not apoptotic cell death. Treatment with ROS scavenger GSH (reduced glutathione), NAC (N-acetyl-l-cysteine) or autophagy inhibitor 3-Methyladenine (3-MA) counteracted daucosterol-induced autophagy and growth inhibition in BGC823 and MCF-7 cancer cells. Daucosterol inhibits cancer cell proliferation by inducing autophagy through ROS-dependent manner and could be potentially developed as an anti-cancer agent.

Oxidative stress responses in watermelon (Citrullus lanatus) as influenced by boron toxicity and drought

This study aimed to investigate the effect of different boron (B) treatments on drought tolerance of watermelon plants. Drought tolerant Citrullus lanatus (Thunb.) Matsum. et Nakai genotype ‘Kar 98’ was grown in controlled greenhouse conditions hydroponically and exposed to drought stress by applying PEG 6000 (polyethylene glycol) in the presence of three boron dosages: 0.05, 0.25 and 1.25 mM. Growth parameters (fresh weight, dry weight and lengths of shoot and roots), leaf relative water content, boron accumulation, lipid peroxidation level and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) enzymes were determined as well as the accumulation of hydrogen peroxide (H2O2) and hydroxyl (•OH)-scavenging activity were assayed. Increasing dosages of boron alone caused more severe growth reduction than combined with PEG 6000-induced drought stress. Induced drought stress caused less accumulation of boron in leaves and roots. B concentration of 1.25 mM caused lipid peroxidation in a reactive oxygen species-independent manner and drought stress-induced lipid peroxidation was alleviated by increasing B dosages. Induced glutathione reductase activity under the combination of 1.25 mM B and PEG 6000-induced drought stress seemed an important physiological response in ‘Kar 98’ plants against multiple stresses.

Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner.

Systematic characterization of the bZIP transcription factor gene family in the rice blast fungus, Magnaporthe oryzae.

Regulatory roles of the basic leucine zipper (bZIP) transcription factors (TFs) in fungi have been identified in diverse cellular processes such as development, nutrient utilization and various stress responses. In this study, the 22 Magnaporthe oryzae genes encoding bZIP TFs were systematically characterized. Phylogenetic analysis of fungal bZIP TFs revealed that seven MobZIPs are Magnaporthe-specific, while others belongs to 15 clades of orthologous Ascomycota genes. Expression patterns of MobZIPs under various conditions showed that they are highly stress responsive. We generated deletion mutants for 13 MobZIPs: nine with orthologues in other fungal species and four Magnaporthe-specific ones. Seven of them exhibited defects in mycelial growth, development and/or pathogenicity. Consistent with the conserved functions of the orthologues, MobZIP22 and MobZIP13 played a role in sulfur metabolism and iron homeostasis respectively. Along with MobZIP22 and MobZIP13, one Magnaporthe-specific gene, MobZIP11 is essential for pathogenicity in a reactive oxygen species-dependent manner. Taken together, our results will contribute to understanding the regulatory mechanisms of the bZIP TF gene family in fungal development, adaptation to environmental stresses and pathogenicity in the rice blast fungus.

PGE2 Promotes BM Hematopoietic Stem Cell Retention Via Stromal Lactate Production­­­­, cAMP and CXCL12/CXCR4 Regulation

The CXCR4/CXCL12 axis is essential for retention and protection from DNA damage of quiescent hematopoietic stem cells (HSC) in their bone marrow (BM) niches. Murine CXCR4+ HSC tightly adhere to BM stromal cells which functionally express cell surface CXCL12. Stress induces secretion of CXCL12 by BM stromal cells and its release to the circulation, mediating hematopoietic stem and progenitor cell (HSPC) egress, recruitment and clinical mobilization. Previously, we reported that Prostaglandin E2 (PGE2), highly produced by COX-2+ BM αSMA+ monocyte/macrophages, upregulates surface CXCR4 expression on enriched human CD34+ HSPC and their CXCL12 induced motility via cAMP activation in vitro. PGE2 inhibits intracellular reactive oxygen species (ROS) generation in HSPC and also increases membrane bound CXCL12 expression by BM stromal cells leading to HSC adhesion to their niche supporting cells in vivo, overall contributing to BM stem cell retention. We also found that elevation in cAMP activation promotes CXCL12 secretion from BM stromal cells, and another report has recently shown that lactate signaling via its major receptor HCA-1 inhibits cAMP.Thus, we hypothesized that the major metabolite lactate, cAMP and PGE2 cross-regulate BM stem cell retention by modulating the CXCR4/CXCL12 axis. We found that both hematopoietic stem cells and BM stromal cells functionally express the lactate receptor HCA-1. Stimulation with PGE2 elevated lactate production by BM stromal cells and stimulation with a HCA-1 receptor agonist, or with lactate, both elevated membrane bound expression of CXCL12 on BM stromal cells. Moreover, since cAMP is elevated by PGE2 signaling whereas lactate signaling was shown to inhibit cAMP, we tested the role of cAMP in CXCL12 expression and secretion by BM stromal cells. We found that in vitro the cAMP enhancer forskolin increased CXCR4 expression by HSPC and in vivo forskolin administration reduced membrane bound CXCL12 levels and elevated CXCL12 secretion as expected. Conversely, in vivo forskolin co-administered with lactate, elevated membrane bound CXCL12 levels and reduced CXCL12 secretion, indicating that lactate limits cAMP elevation and promotes surface CXCL12 expression by BM stromal cells. In accordance,inhibition of cAMP under PGE2 stimulation both in vitro and in vivo, augmented membrane bound CXCL12 expression and inhibited CXCR4 upregulation, mimicking the effects of lactate. We found that PGE2 administration in vivo resulted in reduced CXCR4 expression on primitive BM HSPCs however in vitro PGE2 elevated CXCR4 expression on enriched HSPC. Our results suggest that PGE2 signaling in vivo induces secretion of the metabolite lactate by BM stromal cells, increasing membrane bound CXCL12 expression and reducing expression of CXCR4 on HSPC via cAMP inhibition.Importantly, repeated in vivo administration of PGE2, lactate or its receptor HCA-1 agonist (once daily for 2 days), all reduced CXCR4 expression and steady state egress of HSPC to the bloodcirculation. Thus, PGE2 via downstream lactate secretion acts as a BM stem cell retaining factor. In accordance, we found that in vivo inhibition of PGE2 production by repeated (once daily for five days) injections of COX-2 inhibitors, such as Meloxicam led to HSPC mobilization. This mobilization was abrogated by co-administration of lactate, suggesting that in vivo inhibition of meloxicam induced CXCL12 secretion and release by lactate prevents HSPC mobilization. We found that in vivo COX-2 inhibition reduced membrane expression of CXCL12 by BM stromal cells and elevated surface CXCR4 expression by BM HSPC in a ROS dependent manner. Moreover, neutralization of CXCR4 or CXCL12 by specific antibodies, or ROS by its scavenger NAC, all blocked meloxicam induced stem and progenitor cell mobilization. These results reveal that COX-2 inhibition increased BM CXCL12 secretion and its release to the blood, upregulated CXCR4 leading to HSPC mobilization in a ROS and CXCL12 dependent manner.In conclusion, our results reveal that PGE2 enhances both cAMP elevation and lactate secretion by BM stromal cells in the vicinity of hematopoietic stem cells. Lactate acts in an autocrine manner modulating surface CXCL12 expression by BM niche cells and reduced CXCR4 expression by hematopoietic stem cells via inhibition of cAMP, promoting retention and preservation of hematopoietic stem cells in their BM niches. DisclosuresNo relevant conflicts of interest to declare.

Identification of Hic-5 as a novel scaffold for the MKK4/p54 JNK pathway in the development of abdominal aortic aneurysms.

BackgroundAlthough increased amounts of reactive oxygen species in the pathogenesis of abdominal aortic aneurysm (AAA) are well documented, the precise molecular mechanisms by which reactive oxygen species induce AAAs have not been fully elucidated. This study focused on the role of hydrogen peroxide–inducible clone 5 (Hic‐5), which is induced by hydrogen peroxide and transforming growth factor‐β, in the cellular signaling of AAA pathogenesis.Methods and ResultsUsing the angiotensin II–induced AAA model in Apoe−/− mice, we showed that Apoe−/−Hic‐5−/− mice were completely protected from AAA formation and aortic rupture, whereas Apoe−/− mice were not. These features were similarly observed in smooth muscle cell–specific Hic‐5–deficient mice. Furthermore, angiotensin II treatment induced Hic‐5 expression in a reactive oxygen species–dependent manner in aortic smooth muscle cells in the early stage of AAA development. Mechanistic studies revealed that Hic‐5 interacted specifically with c‐Jun N‐terminal kinase p54 and its upstream regulatory molecule mitogen‐activated protein kinase kinase 4 as a novel scaffold protein, resulting in the expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase 2 activation in aortic smooth muscle cells.ConclusionHic‐5 serves as a novel scaffold protein that specifically activates the mitogen‐activated protein kinase kinase 4/p54 c‐Jun N‐terminal kinase pathway, thereby leading to the induction and activation of matrix metalloproteinases in smooth muscle cells and subsequent AAA formation. Our study provided a novel therapeutic option aimed at inhibiting the mitogen‐activated protein kinase kinase 4–Hic‐5–p54 c‐Jun N‐terminal kinase pathway in the vessel wall, particularly through Hic‐5 inhibition, which may be used to produce more precise and effective therapies.

Treadmill exercise induces neutrophil recruitment into muscle tissue in a reactive oxygen species-dependent manner. An intravital microscopy study.

Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.

Effect of zinc oxide nanomaterials-induced oxidative stress on the p53 pathway

Excessive production of reactive oxygen species (ROS) is a hallmark feature in nanomaterials (NMs) induced cellular toxicity. However, the inter-relationship between NMs induced ROS generation and the cells innate ability to regulate intracellular ROS level in effecting a particular cellular outcome is currently underexplored. Here, using a BJ fibroblast p53 knockdown system, we showed that p53 may be implicated in playing a dual regulatory role to determine cell survivability in response to oxidative stress induced by ZnO NMs. At low level of ZnO NMs induced ROS, p53 triggers expression of antioxidant genes such as SOD2, GPX1, SESN1, SESN2 and ALDH4A1 to restore oxidative homeostasis while at high concentration of ZnO NMs, the elevated level of intracellular ROS activated the apoptotic pathway through p53. The implication of our finding that p53 can function as an important regulator in determining ZnO induced cytotoxicity is highlighted by the differential action of ZnO on p53 deficient and proficient colorectal cell lines. p53 deficient cells cancer cells such as DLD-1 and SW480 are more susceptible to ZnO induced cell death compared to p53 proficient cells such as colon epithelial cells NCM460 and HCT116 cells in a ROS dependent manner. Collectively, our findings showcased a role p53 plays in the context of nanotoxicity and highlights the need to consider the interplay of physicochemical properties of NMs and cell biology.

MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species-dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.

Abstract 535: Shear Stress Upregulates Placental Growth Factor in an in vitro Model of the Vessel Wall in a Reactive Oxygen Species Dependent Manner

Arteriogenesis is the process by which mature arteries form from collateral arterioles after upstream arterial stenosis or occlusion. During collateral remodeling, monocytes are recruited to the vessel wall. Placental growth factor (PLGF) is a potent stimulator of arteriogenesis via monocyte recruitment. Although fluid shear stress is thought to be the primary signal for arteriogenesis, its role in regulating PLGF expression is unknown. However, PLGF is increased in collaterals by upstream arterial ligation. Therefore, we hypothesized that PLGF expression is regulated by shear stress. To test this hypothesis, we created an in vitro model of coronary vessels, consisting of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) cocultured on porous Transwell inserts. HCAEC were then exposed to normal (low) shear stress (LSS) (time average 0.7 dyne/cm2), high shear stress (HSS) (time average 12.4 dyne/cm2), or no shear (static) for 2 hours. HSS significantly increased secreted PLGF protein compared to LSS (~25%, n= 5, p < 0.05) as determined by ELISA. Knockdown of PLGF by siRNA in HCAEC significantly reduced secreted PLGF protein while knockdown in HCASMC had no significant effect, suggesting HCAEC are the primary source of PLGF protein (n=4, p<0.01). Both H2O2 and nitrite (an index of NO production) were significantly increased by shear stress compared to static control (~35%, n= 5, p < 0.05). Treatment with catalase (500 U/mL) attenuated the effects of HSS on PLGF protein levels, whereas L-NAME (100 μM) had no significant effect. Treatment with 5 μM diphenyleneiodonium mimicked the effects of catalase on PLGF protein levels, implicating NADPH oxidase (Nox) as the source of H2O2. PCR analysis revealed Nox-4 as the predominant isoform present in both cell types. Relative Nox-4 mRNA was increased by HSS in HCAEC compared to both LSS and control. Relative gene expression of heme oxygenase-1 (HO-1), which is regulated by Nox-4, was also increased in both cell types by HSS, suggesting the possibility that HO-1 may contribute to the regulation of PLGF expression. These data demonstrate that redox signaling plays a role in the mechanotransduction of shear stress into a regulatory signal for PLGF expression.