Increased Superoxide Production Research Articles

Chloroplast-localized iron superoxide dismutases FSD2 and FSD3 are functionally distinct in Arabidopsis.

Superoxide dismutases (SODs) protect against reactive oxygen species (ROS) by detoxifying superoxide. Three types of SOD are present in plants: FeSOD, CuSOD, and MnSOD. The Arabidopsis thaliana genome contains three FeSOD genes, in which two (FSD2, and FSD3) are targeted to chloroplast thylakoids. Loss of FSD2 or FSD3 expression impairs growth and causes leaf bleaching. FSD2 and FSD3 form heterocomplexes present in chloroplast nucleoids, raising the question of whether FSD2 and FSD3 are functionally interchangeable. In this study, we examined how loss of FSD2 or FSD3 expression affects photosynthetic processes and whether overexpression of one compensates for loss of the other. Whereas loss of the cytosolic FSD1 had little effect, an fsd2 mutant exhibited increased superoxide production, reduced chlorophyll levels, lower PSII efficiency, a lower rate of CO2 assimilation, but elevated non-photochemical quenching (NPQ). In contrast, fsd3 mutants failed to survive beyond the seedling stage and overexpression of FSD2 could not rescue the seedlings. Overexpression of FSD3 in an fsd2 mutant, however, partially reversed the fsd2 mutant phenotype resulting in improved growth characteristics. Overexpression of FSD2 or FSD3, either individually or together, had little effect. These results indicate that, despite functioning as FeSODs, FSD2 and FSD3 are functionally distinct.

Lysophosphatidic Acid Enhances In Vivo Infiltration and Activation of Guinea Pig Eosinophils and Neutrophils via a Rho/Rho-Associated Protein Kinase-Mediated Pathway

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 – 10 μg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

Peptide R18H from BRN2 Transcription Factor POU Domain Displays Antitumor Activity In Vitro and In Vivo and Induces Apoptosis in B16F10-Nex2 Cells.

BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor. To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells. Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment. Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H. Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo.

Synergistic Role of Oxidative Stress and Blood-Brain Barrier Permeability as Injury Mechanisms in the Acute Pathophysiology of Blast-induced Neurotrauma

Blast-induced traumatic brain injury (bTBI) has been recognized as the common mode of neurotrauma amongst military and civilian personnel due to an increased insurgent activity domestically and abroad. Previous studies from our laboratory have identified enhanced blood-brain barrier (BBB) permeability as a significant, sub-acute (four hours post-blast) pathological change in bTBI. We also found that NADPH oxidase (NOX)-mediated oxidative stress occurs at the same time post-blast when the BBB permeability changes. We therefore hypothesized that oxidative stress is a major causative factor in the BBB breakdown in the sub-acute stages. This work therefore examined the role of NOX1 and its downstream effects on BBB permeability in the frontal cortex (a region previously shown to be the most vulnerable) immediately and four hours post-blast exposure. Rats were injured by primary blast waves in a compressed gas-driven shock tube at 180 kPa and the BBB integrity was assessed by extravasation of Evans blue and changes in tight junction proteins (TJPs) as well as translocation of macromolecules from blood to brain and vice versa. NOX1 abundance was also assessed in neurovascular endothelial cells. Blast injury resulted in increased extravasation and reduced levels of TJPs in tissues consistent with our previous observations. NOX1 levels were significantly increased in endothelial cells followed by increased superoxide production within 4 hours of blast. Blast injury also increased the levels/activation of matrix metalloproteinase 3 and 9. To test the role of oxidative stress, rats were administered apocynin, which is known to inhibit the assembly of NOX subunits and arrests its function. We found apocynin completely inhibited dye extravasation as well as restored TJP levels to that of controls and reduced matrix metalloproteinase activation in the sub-acute stages following blast. Together these data strongly suggest that NOX-mediated oxidative stress contributes to enhanced BBB permeability in bTBI through a pathway involving increased matrix metalloproteinase activation.

Aggravated behavioral and neurochemical deficits and redox imbalance in mice with enhanced neonatal iron intake: improvement by biochanin A and role of microglial p38 activation

ABSTRACT Objectives: We aim to investigate the joint effect of iron (enhanced neonatal iron intake), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and biochanin A (BA, oral administration) and possible mechanisms for action on behavioral and neurochemical indicators in the mice. Methods: Rotarod test, pole test and swim test were used to evaluate animal behavior. The neurochemical analysis was conducted by HPLC-ECD. Oxidative stress was determined in this study. Further mechanism was investigated through in vitro experiments. Results: Iron and MPTP co-administration significantly induced behavioral deficits and decreased striatal dopamine content in the male and female mice. The co-administration of iron and MPTP also significantly induced redox imbalance in the substantia nigra (SN) of mice. Furthermore, BA significantly improved behavioral deficits and increased striatal dopamine content in the mice co-treated with iron and MPTP. BA also significantly improved redox imbalance in the SN of mice co-administered with iron and MPTP. Finally, we showed that iron and 1-Methyl-4-phenylpyridinium (MPP+) co-treatment significantly increased superoxide production in microglial cultures by inducing p38 mitogen-activated protein kinase (MAPK) activation. BA also significantly decreased superoxide production and p38 MAPK phosphorylation in the cultures co-treated with iron and MPP+. Conclusion: Iron and MPTP co-treatment may result in worsened behavioral and neurochemical deficits and aggravated redox imbalance through inducing microglial p38 MAPK activation. BA may improve behavioral and neurochemical deficits and redox imbalance through repressing microglial p38 MAPK activation.

An SOD1 deficiency aggravates proteasome inhibitor bortezomib-induced testicular damage in mice

Proteasomes play a key role in maintaining cellular homeostasis by the proteolytic removal of proteins, including ubiquitinated proteins and/or oxidatively-damaged proteins. The proteasome inhibitor bortezomib (BTZ) has been reported to exert testicular toxicity in mice. In the current study, we treated SOD1-knockout (KO) mice with BTZ and investigated the issue of whether oxidative stress is involved in the development of testicular toxicity. The BTZ treatment significantly increased superoxide production and cell death in the testes of SOD1-KO mice compared to wild-type (WT) mice. We also found that high levels of both ubiquitinated proteins and p62 accumulated and underwent aggregation in the seminiferous tubules of BTZ-injected SOD1-KO mice. Furthermore, the proteolytic activities of proteasomes were significantly decreased in the testes of BTZ-injected SOD1-KO mice compared to their WT counterparts. These results suggest that a combination of oxidative stress caused by an SOD1 deficiency and proteasome inhibition by BTZ accelerates the impairment of proteasomes, which results in severe testicular damage in SOD1-KO mice.

1,2,3,4,6-Penta-O-galloyl-β-d-glucose modulates perivascular inflammation and prevents vascular dysfunction in angiotensin II-induced hypertension.

Background and PurposeHypertension is a multifactorial disease, manifested by vascular dysfunction, increased superoxide production, and perivascular inflammation. In this study, we have hypothesized that 1,2,3,4,6‐penta‐O‐galloyl‐β‐d‐glucose (PGG) would inhibit vascular inflammation and protect from vascular dysfunction in an experimental model of hypertension.Experimental ApproachPGG was administered to mice every 2 days at a dose of 10 mg·kg−1 i.p during 14 days of Ang II infusion. It was used at a final concentration of 20 μM for in vitro studies in cultured cells.Key ResultsAng II administration increased leukocyte and T‐cell content in perivascular adipose tissue (pVAT), and administration of PGG significantly decreased total leukocyte and T‐cell infiltration in pVAT. This effect was observed in relation to all T‐cell subsets. PGG also decreased the content of T‐cells bearing CD25, CCR5, and CD44 receptors and the expression of both monocyte chemoattractant protein 1 (CCL2) in aorta and RANTES (CCL5) in pVAT. PGG administration decreased the content of TNF+ and IFN‐γ+ CD8 T‐cells and IL‐17A+ CD4+ and CD3+CD4−CD8− cells. Importantly, these effects of PGG were associated with improved vascular function and decreased ROS production in the aortas of Ang II‐infused animals independently of the BP increase. Mechanistically, PGG (20 μM) directly inhibited CD25 and CCR5 expression in cultured T‐cells. It also decreased the content of IFN‐γ+ CD8+ and CD3+CD4−CD8− cells and IL‐17A+ CD3+CD4−CD8− cells.Conclusion and ImplicationPGG may constitute an interesting immunomodulating strategy in the regulation of vascular dysfunction and hypertension.Linked ArticlesThis article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc

Increased circulating trimethylamine N-oxide plays a contributory role in the development of endothelial dysfunction and hypertension in the RUPP rat model of preeclampsia

ABSTRACTObjective: We examined whether trimethylamine-N-oxide (TMAO) plays a role in endothelial dysfunction and hypertension in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia (PE). Methods: Normal pregnant rats and RUPP rats were treated without or with 3,3-Dimethyl-1-butanol (DMB, a TMAO inhibitor) from gestational day 14. Results: On day 19 of gestation, RUPP rats had higher plasma TMAO, impaired vasodilation and hypertension, decreased interleukin (IL)-10, increased superoxide production and proinflammatory cytokines in the aorta. All of which were reversed by DMD. Conclusion: Increased circulating TMAO downregulates IL-10 and promotes vascular inflammation and oxidative stress, contributing to endothelial dysfunction and hypertension in PE.

The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H₂O₂ levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H₂O₂ production. H₂O₂ metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H₂O₂. Stat5b silencing finally reduces AKT expression, a prosurvival signal. Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H₂O₂ accumulation and the ensuing oxidative stress enhances β-cell damage.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-β, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

Mitochondrial LonP1 protects cardiomyocytes from ischemia/reperfusion injury in vivo

RationaleLonP1 is an essential mitochondrial protease, which is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. However, the importance of LonP1 during cardiac stress is largely unknown. ObjectiveTo determine the functions of LonP1 during ischemia/reperfusion (I/R) injury in vivo, and hypoxia-reoxygenation (H/R) stress in vitro. Methods and resultsLonP1 was induced 2-fold in wild-type mice during cardiac ischemic preconditioning (IPC), which protected the heart against ischemia-reperfusion (I/R) injury. In contrast, haploinsufficiency of LonP1 (LONP1+/−) abrogated IPC-mediated cardioprotection. Furthermore, LONP1+/− mice showed significantly increased infarct size after I/R injury, whereas mice with 3–4 fold cardiac-specific overexpression of LonP1 (LonTg) had substantially smaller infarct size and reduced apoptosis compared to wild-type controls. To investigate the mechanisms underlying cardioprotection, LonTg mice were subjected to ischemia (45 min) followed by short intervals of reperfusion (10, 30, 120 min). During early reperfusion, the left ventricles of LonTg mice showed substantially reduced oxidative protein damage, maintained mitochondrial redox homeostasis, and showed a marked downregulation of both Complex I protein level and activity in contrast to NTg mice. Conversely, when LonP1 was knocked down in isolated neonatal rat ventricular myocytes (NRVMs), an up-regulation of Complex I subunits and electron transport chain (ETC) activities was observed, which was associated with increased superoxide production and reduced respiratory efficiency. The knockdown of LonP1 in NRVMs caused a striking dysmorphology of the mitochondrial inner membrane, mitochondrial hyperpolarization and increased hypoxia-reoxygenation (H/R)-activated apoptosis. Whereas, LonP1 overexpression blocked H/R-induced cell death. ConclusionsLonP1 is an endogenous mediator of cardioprotection. Our findings show that upregulation of LonP1 mitigates cardiac injury by preventing oxidative damage of proteins and lipids, preserving mitochondrial redox balance and reprogramming bioenergetics by reducing Complex I content and activity. Mechanisms that promote the upregulation of LonP1 could be beneficial in protecting the myocardium from cardiac stress and limiting I/R injury.

The Effects of Environmental Stressors on Aggregation in <i>Parachlorella kessleri</i>

The induction of biofilm formation has been explored as a means of harvesting microalgae for bioprocessing applications. Environmental stressors have been implicated in the induction of biofilm formation; however, it is unclear whether all stressors, or a select few, are responsible. This study aimed to investigate the effects of three stressors, nitrogen depletion, reduced or elongated day lengths, and increased culture turbulence on bio-film formation of Parachlorella kessleri. We also examined corresponding effects on growth and production of reactive oxygen species. Turbulence induced the greatest response in which a significant decrease in growth plus an increase in superoxide production and flocculation efficiency were seen for the 300-rpm treatment. For varying day lengths, stress response was not observed, however, a significant increase in EPS secretion was measured in both shorter and longer days. Nitrogen depletion induced a low-level stress response, in which superoxide production increased for the highest concentrations, while growth was not impacted. In contrast to previous studies on nitrogen depletion, a significant increase in EPS secretion was not observed. Results indicate that stress response varies according to type and magnitude. EPS production and thus biofilm formation are not linked to the stress indicators investigated. P. kessleri uses small quantities of EPS to contribute to cell stickiness, but not necessarily to the full formation of a biofilm; however, cell stickiness served as a mechanism for substrate adherence and cellular aggregation none the less.

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines.

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L-cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G 0 and G 2 phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.

Developmental effects of trichloroacetate in Zebrafish embryos: Association with the production of superoxide anion.

To assess the developmental toxicity of trichloroacetate (TCA), zebrafish embryos were exposed to 8 to 48 mM of TCA and evaluated for developmental milestones from 8- to 144-hour postfertilization (hpf). All developmental toxicities are reported in this paper. Embryos were found to have developed edema in response to 16 to 48 mM of TCA exposure at 32- to 80-hpf, experienced delay in hatching success in response to 24 to 48 mM at 80-hpf. Lordosis was observed in developing embryos exposed to 40 to 48 mM at 55- to 144-hpf. The observed toxic effects of TCA exposure were found to be concentration and exposure period independent. Effects were found to be associated with increases in superoxide anion production, but these increases were also found to be concentration and time independent. TCA resulted in concentration-dependent increases in embryonic lethality at 144-hpf, with an LC50 determined to be 29.7 mM.

Coronary Microvascular and Cardiac Dysfunction Due to Homocysteine Pathometabolism; A Complex Therapeutic Design.

In various metabolic diseases, both the coronary circulation and cardiac metabolism are altered. Here we summarize the effects of a condition called hyperhomocysteinemia (HHcy) - which can develop due to genetic and/or environmental causes - on the function of coronary microvessels and heart. This metabolic disease is underappreciated, yet even mild or moderate elevation of plasma concentrations of homocystein (Hcy, plasma Hcy >16 µM), a sulfur-containing amino acid produced via methionine metabolism) leads to coronary and peripheral artery and even venous vessel diseases, eliciting vasomotor dysfunction and increased thrombosis, consequently increased morbidity and mortality. Yet the underlying mechanisms have not yet been revealed. Recent studies indicated that there are common pathomechanisms, which may affect several cellular functions. With methionin diet-induced HHcy two main pathomechanisms were revealed: the dysfunction of nitric oxide (NO) pathway resulting in reduced dilator responses of arteries and arterioles, and the simultaneously increased thromboxane A2 (TXA2) activity both in vessels and platelets. These changes are likely due to an increased production of reactive oxidative species (oxidative stress) due to increased NADPH oxidase assembly, which eventually lead to inflammatory processes (indicated by increases in TNFα, NFκbeta, p22phox, p67phox, and rac-1, levels) and changes in various gene expressions and morphological remodeling of vessels. Increased superoxide production and reduced availability of NO alter the regulation of mitochondrial function in the myocardium. The interactions of these pathomechanisms may explain why HHcy increases the uptake of glucose and lactate and decreases the uptake of free fatty acid by the heart. The pathological consequences of HHcy could be worsening by the simultaneous presence of other risk factors, such as hyperlipidemia, diabetes mellitus and metabolic syndrome. All in all, HHcy and associated pathometabolism lead to severe changes and dysfunctions of coronary arterial vessels and cardiac function, which may not always be apparent in clinical settings but most likely contribute to the increased prevalence of cardiovascular diseases and mortality, which however can be reduced by appropriate prevention and treatments. We believe that HHcy is an underestimated - likely due to inappropriate clinical trials - but serious disease condition because it promotes the development of atherosclerosis in large arterial vessels, vasomotor dysfunction in microvessels, hypertension and thrombosis. In this review, we will summarize previous functional findings focusing on coronary vessels and cardiac function and the underlying cellular and molecular mechanisms enabling the development of novel treatments.

Understanding the Role of Surface Charge in Cellular Uptake and X-ray-Induced ROS Enhancing of Au-Fe3O4 Nanoheterodimers.

Au-Fe3O4 nanoheterodimers were obtained by thermally decomposing iron oleate on presynthesized gold nanoparticles. Water solubility as well as surface charges were achieved by encapsulating the initially hydrophobic Au-Fe3O4 nanoheterodimers in a self-assembled bilayer shell formed either by 1-octadecylpyridinium, providing positive surface charges, or by 4-dodecylbenzenesulfonate, yielding a negatively charged surface. The surface charge and surface architecture were shown to control both the cellular entry and the intracellular trafficking of the Au-Fe3O4 nanoheterodimers. The positively charged (1-octylpyridinium-terminated) Au-Fe3O4 nanoheterodimers were internalized by both breast cancer cells (MCF-7) and epithelial cells (MCF-10 A), wherein they were electrostatically bound at the negatively charged membranes of the cell organelles and, in particular, adsorbed onto the mitochondrial membrane. The treatment of MCF-7 and MCF-10 cells with a fractional X-radiation dose of 1 Gy resulted into a large increase of superoxide production, which arose from the Au-Fe3O4 nanoheterodimer-induced mitochondrial depolarization. In contrast, the negatively charged (4-dodecylbenzenesulfonate-terminated) Au-Fe3O4 nanoheterodimers preferentially invaded the cancerous MCF-7 cells by direct permeation. X-ray treatment of MCF-7 cells, loaded with anionic Au-Fe3O4 nanoheterodimers, yielded the increase of both hydroxyl radical and cytoplasmic superoxide formation. The X-radiation-induced activation of the Fe3O4 surfaces, consisting of Fe2+ and Fe3+ cations, triggered the catalysis of the hydroxyl radical production, whereas superoxide formation presumably occurred through X-ray-induced photoelectron emission near the Au surface. Since hydroxyl radicals are highly cytotoxic and the negatively charged Au-Fe3O4 NHDs spare the healthy MCF-10A cells, these Au-Fe3O4 nanoheterodimers exhibit a higher potential for radiation therapy than the positively charged Au-Fe3O4 nanoheterodimers. Encouraging results from the clonogenic cell survival assay and DMF calculations corroborate the excellent performance of the anionic Au-Fe3O4 nanoheterodimers as an X-ray dosage enhancer.

Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts.

Increasing evidence highlights that senescence plays an important role in idiopathic pulmonary fibrosis (IPF). This study delineates the specific contribution of mitochondria and the superoxide they form to the senescent phenotype of lung fibroblasts from IPF patients (IPF‐LFs). Primary cultures of IPF‐LFs exhibited an intensified DNA damage response (DDR) and were more senescent than age‐matched fibroblasts from control donors (Ctrl‐LFs). Furthermore, IPF‐LFs exhibited mitochondrial dysfunction, exemplified by increases in mitochondrial superoxide, DNA, stress and activation of mTORC1. The DNA damaging agent etoposide elicited a DDR and augmented senescence in Ctrl‐LFs, which were accompanied by disturbances in mitochondrial homoeostasis including heightened superoxide production. However, etoposide had no effect on IPF‐LFs. Mitochondrial perturbation by rotenone involving sharp increases in superoxide production also evoked a DDR and senescence in Ctrl‐LFs, but not IPF‐LFs. Inhibition of mTORC1, antioxidant treatment and a mitochondrial targeting antioxidant decelerated IPF‐LF senescence and/or attenuated pharmacologically induced Ctrl‐LF senescence. In conclusion, increased superoxide production by dysfunctional mitochondria reinforces lung fibroblast senescence via prolongation of the DDR. As part of an auto‐amplifying loop, mTORC1 is activated, altering mitochondrial homoeostasis and increasing superoxide production. Deeper understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.

Curcumin prevents potassium dichromate (K2Cr2O7)-induced renal hypoxia

Curcumin exhibits several therapeutic properties. Potassium dichromate (K2Cr2O7)-induced nephropathy is associated with oxidative stress. Reactive oxygen species production affects renal oxygenation that may participate in the progression of renal damage. The aim of the present work was to elucidate whether K2Cr2O7-induced nephropathy is associated to partial O2 pressure (pO2) impairment and if curcumin is able to prevent it. Four groups of rats were studied: control group; K2Cr2O7 group (12.5 mg/kg, s.c.); curcumin + K2Cr2O7 group, in which animals were treated with curcumin (400 mg/kg/day, p.o.) for 10 days before K2Cr2O7 injection; and curcumin group. All animals were sacrificed 48 h after the end of the treatments. K2Cr2O7 administration increased renal function markers and decreased glomerular filtration rate, pO2 and renal perfusion. Concerning hemodynamic parameters, K2Cr2O7 increased mean arterial pressure and renal vascular resistance and reduced renal blood flow. The hemodynamic changes were attributed to decreased availability of nitric oxide and increased 3-nitrotyrosine levels. Moreover, increased superoxide anion production and vascular endothelial growth factor levels were observed after K2Cr2O7 administration. Curcumin attenuated all the above-described alterations. Our results suggest that the protective effects of curcumin in K2Cr2O7-induced nephropathy are associated with its ability to prevent O2 supply reduction.

Abstract P332: Hesperidin in Citrus Fruit Juice Plays a Role in Preventing Cognitive Impairment Induced by Ischemic Brain Damage

Recently we reported that drinking Citrus iyo juice (CI) inhibited more effectively vascular remodeling in the inflammation-induced vascular injury mouse model than Citrus unshiu juice (CU). These results led us to explore the possibility that citrus fruits juice drinking could attenuate cognitive decline in transient cerebral ischemia mouse model, focusing on the effects of flavanone, hesperidin, which is more abundantly contained in CI compared with CU and has antioxidant activity. Eight-week-old male C57BL/6 mice were administrated 10 % CI or CU in drinking water or 100 mg/kg/day hesperidin orally by gavage. Two weeks after administration, brain ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 18 minutes. The Y maze task was performed 2 weeks after BCCAO operation. After cognitive task, cerebral blood flow (CBF) was measured by a laser speckle flowmetry. Morphological changes in the hippocampus were examined. Administration of CI, CU or hesperidin did not influence systolic blood pressure, body weight and brain weight. Cognitive function was significantly impaired (sham, 71% (16 of 23) vs BCCAO, 55% (14 of 27) in Y maze) with the increase in superoxide anion production after BCCAO. The cognitive impairment was more effectively attenuated by the administration of CI than CU (CI, 66% (13 of 23); CU, 61% (17 of 27)) with the significant increase in CBF. Interestingly, we also observed that the treatment with hesperidin significantly prevented cognitive decline (67% (13 of 21)) after BCCAO. The increase in superoxide anion production 24 hours after BCCAO (expressed as fold-increase compared to sham) was attenuated by CI or hesperidin, not by CU (BCCAO, 4.0; CI, 1.2; hesperidin, 1.1; CU, 3.0). The fold-increase in TNF-α mRNA in the hippocampus 24 hours after BCCAO was prevented by hesperidin (BCCAO 5.0; hesperidin, 2.8). Cell number in CA1 region of hippocampus decreased in BCCAO-operated mice. Hesperidin treatment attenuated this decrease (sham, 163±5; BCCAO, 136±4; hesperidin, 154±4). These results suggest that the intake of hesperidin in citrus fruits juice should prevent cognitive decline after brain ischemia at least in part due to reduction of oxidative stress, inflammatory cytokine and an increase in CBF.