Manoalide Shows Mutual Interaction between Cellular and Mitochondrial Reactive Species with Apoptosis in Oral Cancer Cells.

We previously found that marine sponge-derived manoalide induced antiproliferation and apoptosis of oral cancer cells as well as reactive species generations probed by dichloro-dihydrofluorescein diacetate (DCFH-DA) and MitoSOX Red. However, the sources of cellular and mitochondrial redox stresses and the mutual interacting effects between these redox stresses and apoptosis remain unclear. To address this issue, we examined a panel of reactive species and used the inhibitors of cellular reactive species (N-acetylcysteine (NAC)), mitochondrial reactive species (MitoTEMPO), and apoptosis (Z-VAD-FMK; ZVAD) to explore their interactions in manoalide-treated oral cancer Ca9-22 and CAL 27 cells. Hydroxyl (˙OH), nitrogen dioxide (NO2˙), nitric oxide (˙NO), carbonate radical-anion (CO3 ˙-), peroxynitrite (ONOO-), and superoxide (O2 ˙-) were increased in oral cancer cells following manoalide treatments in terms of fluorescence staining and flow cytometry. Cellular reactive species (˙OH, NO2 ·, ˙NO, CO3 ˙-, and ONOO-) as well as cellular and mitochondrial reactive species (O2 ˙-) were induced in oral cancer cells following manoalide treatment for 6 h. NAC, MitoTEMPO, and ZVAD inhibit manoalide-induced apoptosis in terms of annexin V and pancaspase activity assays. Moreover, NAC inhibits mitochondrial reactive species and MitoTEMPO inhibits cellular reactive species, suggesting that cellular and mitochondrial reactive species can crosstalk to regulate each other. ZVAD shows suppressing effects on the generation of both cellular and mitochondrial reactive species. In conclusion, manoalide induces reciprocally activation between cellular and mitochondrial reactive species and apoptosis in oral cancer cells.

Delphinidin Increases the Sensitivity of Ovarian Cancer Cell Lines to 3-Bromopyruvate.

3-Bromopyruvic acid (3-BP) is a promising anticancer compound. Two ovary cancer (OC) cell lines, PEO1 and SKOV3, showed relatively high sensitivity to 3-BP (half maximal inhibitory concentration (IC50) of 18.7 and 40.5 µM, respectively). However, the further sensitization of OC cells to 3-BP would be desirable. Delphinidin (D) has been reported to be cytotoxic for cancer cell lines. We found that D was the most toxic for PEO1 and SKOV3 cells from among several flavonoids tested. The combined action of 3-BP and D was mostly synergistic in PEO1 cells and mostly weakly antagonistic in SKOV3 cells. The viability of MRC-5 fibroblasts was not affected by both compounds at concentrations of up to 100 µM. The combined action of 3-BP and D decreased the level of ATP and of dihydroethidium (DHE)-detectable reactive oxygen species (ROS), cellular mobility and cell staining with phalloidin and Mitotracker Red in both cell lines but increased the 2',7'-dichlorofluorescein (DCFDA)-detectable ROS level and decreased the mitochondrial membrane potential and mitochondrial mass only in PEO1 cells. The glutathione level was increased by 3-BP+D only in SKOV3 cells. These differences may contribute to the lower sensitivity of SKOV3 cells to 3-BP+D. Our results point to the possibility of sensitization of at least some OC cells to 3-BP by D.

Increasing DNA binding affinity of doxorubicin by loading on Fe3O4 nanoparticles: A multi-spectroscopic study.

Magnetic Fe3O4 nanoparticles were synthesized successfully by co-precipitation method and characterized using XRD, SEM and EDS analyses. Then doxorubicin (DOX, a known anticancer drug) was loaded onto nanoparticles. In vitro DNA interaction of free DOX and loaded DOX onto Fe3O4 nanoparticles (DOX-Fe3O4) was investigated by DNA-viscosity measurements, UV-visible and fluorescence spectroscopies. The obtained values for binding constant of DOX and DOX-Fe3O4 compounds from UV-visible spectroscopies were 0.04 × 105 and 0.68 × 105 L mol-1, respectively, which confirms DOX-Fe3O4 compound have a stronger interaction with CT-DNA compared to DOX. Considerable changes on viscosity of the compounds recommended that their binding mode with CT-DNA is intercalative binding. Fluorescence intensity of DOX and DOX-Fe3O4 was quenched via static process by regular addition of CT-DNA. Thermodynamic parameters suggest that Van der Waals forces and hydrogen bonding for DOX and electrostatic forces for DOX-Fe3O4 are predominantly responsible for interaction with CT-DNA. Competition fluorescence studies were done by Hoechst 33258 as a well-known groove binder and ethidium bromide (EtBr) as a known intercalator probe. Percentage of displacement for EtBr-DNA complex with DOX and DOX-Fe3O4 was 39% and 61%, and for Hoechst-DNA complex was 9% and 5%, respectively. These results confirmed that both compounds are intercalator binders, although DOX-Fe3O4 with a further 22% displacement is a stronger intercalator binder than DOX. The stronger interaction of DOX-Fe3O4 compared to DOX suggests that the current system can be used as a new and effective way to targeted therapy of anticancer drugs.

Comparison of three analytical methods for superoxide produced by activated immune cells.

Superoxide plays a key role in normal immune function and inflammatory diseases. In order to evaluate normal immune function or screen inhibitors of superoxide production for treating inflammatory diseases, it is very important to detect superoxide with good accuracy, sensitivity, and flexibility. In present study, we investigated three analysis methods of superoxide, colorimetric assay by WST-8, fluorescence assay by dihydroethidium and chemiluminescence assay by lucigenin, compared their precisions, specificities, sensitivities and time curve characteristics in superoxide analysis, and then validate their values in the screening of anti-inflammatory compounds. The results reveal that three analysis methods of superoxide all have good precisions and high specificities but have different sensitivities and time curve characteristics, which suggest their different applications. In addition, they can all be used in the screening of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors and anti-inflammatory compounds.

Association of Superoxide-Induced Oxidative Stress With Rotator Cuff Tears in Human Patients.

Rotator cuff degeneration is one of the factors contributing to rotator cuff tears. Oxidative stress is involved in tendon degeneration, and superoxide-induced oxidative stress plays a pathological role in regulating the balance between oxidation and reduction. The role of oxidative stress in rotator cuff tears, however, is unclear. This study, therefore, aimed to investigate the contribution of superoxide-induced oxidative stress to rotator cuff tears. Seventy patients were recruited and divided into two groups: patients with (Ruptured group) and those without (Unruptured group) a rotator cuff tear. Specimens from both groups were collected during surgery. Degeneration morphology was classified according to the degeneration score. Superoxide-induced oxidative stress was assessed according to dihydroethidium (DHE) relative fluorescence intensity, capacity for antioxidation according to superoxide dismutase (SOD) activity, and the balance between oxidation and reduction based on the redox ratio. Data were compared between groups. Correlations between the degeneration score and the oxidative stress factors were calculated. Degeneration score and DHE relative fluorescence intensity were significantly higher in the Ruptured than the Unruptured group. The SOD activity was not significantly different between groups. Degeneration score was positively correlated with both DHE relative fluorescence intensity and SOD activity. Thus, superoxide-induced oxidative stress and tendon degeneration were greater in rotator cuff tear tissues than in those with no tear, suggesting that oxidative imbalance may be involved in degenerative rotator cuff tears. Clinical Relevance: Understanding the mechanisms of superoxide-induced oxidative stress may lead to targeted tissue therapy for degenerative rotator cuff tears. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:212-218, 2020.

A palladium(II) complex with the Schiff base 4-chloro-2-(N-ethyliminomethyl)-phenol: Synthesis, structural characterization, and in vitro and in silico biological activity studies.

The synthesis and characterization of the Pd(II) complex of the formula [Pd(L)2] 1 with the Schiff base 4-chloro-2-(N-ethyliminomethyl)-phenol (HL) as derived in situ via the condensation reaction of 5-chloro-salicylaldehyde and ethylamine was undertaken. The structure of 1 was verified by single-crystal X-ray crystallography. The ability of 1 to interact with calf-thymus (CT) DNA was studied by UV-vis and viscosity experiments, and its ability to displace ethidium bromide (EB) from the DNA-EB conjugate was revealed by fluorescence spectroscopy. It was found that intercalation is the most possible mode of interaction with CT DNA. Additionally, DNA electrophoretic mobility experiments showed that 1 interacts with the plasmid pBluescript SK(+) (pDNA) as proved by the formation of unusual mobility DNA bands and degradation of relaxed pDNA at concentration of 5 mM. The interaction of 1 with human (HSA) and bovine serum albumin (BSA) was monitored revealing its reversible binding to albumins. The complex showed noteworthy antimicrobial activity against one (Bacillus subtilis) of the five tested bacteria. In order to explain the described in vitro activity of the compound, we adopted molecular docking studies on the crystal structure of HSA, BSA, CT DNA and DNA-gyrase. Furthermore, in silico predictive tools have been employed to study the properties of the complex. The in silico studies are adopted on a multitude of proteins involved in cancer growth, as well as prediction of drug-induced changes of gene expression profile, protein- and mRNA-based prediction results, prediction of sites of metabolism, cytotoxicity for cancer cell lines, etc.

Multifunctional titanium phosphate nanoparticles for site-specific drug delivery and real-time therapeutic efficacy evaluation.

Receptor-targeted delivery systems have been proposed as means of concentrating therapeutic agents to improve therapeutic effects on disease sites and reduce side effects on normal issues. Herein, we synthesized biocompatible folic acid (FA)-functionalized DHE-modified TiP (TiP-PAH-DHE-FA) nanoparticles as a drug delivery system that possessed high drug loading capability and enhanced folate-receptor-mediated cellular uptake. Moreover, it also allowed drug effect evaluation based on the real-time monitoring of the fluorescence intensity of HE molecules that are triggered by intercellular ROS. This acquired drug delivery system provided a novel platform to integrate efficient cell-specific drug delivery with real-time monitoring of therapeutic efficacy.

Spectral and thermochemical research of the DNA polyplex with chitosan formation process and the influence of anionic and cationic compounds.

In this paper, the results of a spectral and thermochemical study of the DNA polyplex formation with chitosan and the effect of ethidium bromide polyplexes, sodium dodecyl sulfate, n-octyltrimethyl ammonium bromide, poly(4-styrenesulfonic acid), and heparin on the stability of the complexes are considered. It has been established that chitosan forms thermodynamically stable complexes with ethidium bromide (EtBr), in which there exists one monomer unit of chitosan for two ethidium bromide ones. The interaction of ethidium bromide with chitosan leads to a charge exchange of the polymer surface. The impact of chitosan on the intercalated DNA-EtBr complex conditions a release of EtBr with a polyplex formation. The process of polyplex formation in the presence of ethidium bromide proceeds endothermically, and in its absence the reaction is exothermic. The polyplex particles formed from DNA after release of EtBr are larger and have a smaller charge, as compared to the polyplex particles obtained without ethidium bromide. It has been found that anionic compounds cause the degradation of polyplexes, and it can prove to be a significant obstacle for using chitosan polyplexes in transfection, since in the presence of heparin in the bloodstream, the complexes will break down before reaching the target.

Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis, characterization, antitumor activities and rich DNA/HSA interaction studies.

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV-Vis, 1H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of -NH2 and the oxygen of -COO- groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K562) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide-DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA. Communicated by Ramaswamy H. Sarma.

A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide ß.

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 µg/ml CRP and 30 µM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid ß (Aß) 1-42 peptide induced superoxide anion formation in a concentration-dependent manner. Aß peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aß 1-42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 µM arachidonic acid. The inhibition of NOXs by 10 µM VAS2870 abolished Aß-dependent potentiation of platelet aggregation in response to 10 µM arachidonic acid, suggesting that the pro-thrombotic activity of Aß peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aß peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets.

New monofunctional platinum(II) and palladium(II) complexes: Studies of the nucleophilic substitution reactions, DNA/BSA interaction, and cytotoxic activity.

Four new complexes [Pd(H2LtBu)Cl]Cl (Pd1), [Pt(H2LtBu)Cl]Cl (Pt1), [Pd(Me2LtBu)Cl]Cl (Pd2) and [Pt(Me2LtBu)Cl]Cl (Pt2) (where H2LtBu = 2,6-bis(5-(tert-butyl)-1H-pyrazol-3-yl)pyridine and Me2LtBu = 2,6-bis(5-(tert-butyl)-1-methyl-1H-pyrazol-3-yl)pyridine) were synthesized and characterized by elemental microanalysis, IR, 1H NMR and ESI-MS methods. The reactivity of complexes towards thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys) and guanosine-5'-monophosphate (5'-GMP) was investigated. The obtained order was established as follows: Tu > l-Cys > l-Met > 5'-GMP. Complexes Pd1 and Pt1, that contain H2LtBu as chelator, showed higher reactivity towards biomolecules than those with Me2LtBu. The interaction of complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was studied by UV-Vis and fluorescence spectroscopy. The results have shown that complexes can bind to DNA exhibiting high binding constants (Kb = 104 M-1). Obtained results during the examination of competitive reaction with ethidium bromide (EB) showed that complexes can replace EB-bound DNA. High values of binding constants indicate good binding affinity of complexes towards BSA. We evaluated the stability differences between complexes based on terpy as well as H2LtBu/Me2LtBu by DFT calculations (B3LYP(CPCM)/LANL2DZp), showing that both tridentate ligand systems lead to complexes of similar stability. The results of biological testing showed that all complexes exert moderate to high selective cytotoxicity, inducing apoptosis and autophagy in HeLa and PANC-1 tumor cell lines. Pd1 exhibited the strongest cytotoxic effect. Finally, cell cycle analysis showed that in HeLa cells Pd1, Pd2 and Pt1 induced accumulation of cells in S phase, whereas in PANC-1 cells Pd2 and Pt1 induced G2/M cycle arrest and Pd1 induced G0/G1 arrest.

Fluidic shear stress increases the anti-cancer effects of ROS-generating drugs in circulating tumor cells.

PURPOSE: Many anti-cancer drugs are used in chemotherapy; however, little is known about their efficacy against circulating tumor cells (CTCs). In this study, we investigated whether the pulsatile fluidic shear stress (SS) in human arteries can affect the efficacy of anti-cancer drugs. METHODS: Cancer cells were circulated in our microfluidic circulatory system, and their responses to drug and SS treatments were determined using various assays. Breast and cervical cancer cells that stably expressed apoptotic sensor proteins were used to determine apoptosis in real-time by fluorescence resonance energy transfer (FRET)-based imaging microscopy. The occurrence of cell death in non-sensor cells were revealed by annexin V and propidium iodide staining. Cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS) levels were determined by staining cells with two ROS-detecting dyes: 2',7'-dichlorofluorescin diacetate and dihydroethidium. RESULTS: Fluidic SS significantly increased the potency of the ROS-generating drugs doxorubicin (DOX) and cisplatin but had little effect on the non-ROS-generating drugs Taxol and etoposide. Co-treatment with SS and ROS-generating drugs dramatically elevated ROS levels in CTCs, while the addition of antioxidants abolished the pro-apoptotic effects of DOX and cisplatin. More importantly, the synergistic killing effects of SS and DOX or cisplatin were confirmed in circulated lung, breast, and cervical cancer cells, some of which have a strong metastatic ability. CONCLUSIONS: These findings suggest that ROS-generating drugs are more potent than non-ROS-generating drugs for destroying CTCs under pulsatile fluidic conditions present in the bloodstream. This new information is highly valuable for developing novel therapies to eradicate CTCs in the circulation and prevent metastasis.

Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini "Singlet Oxygen Generator" (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen (1O2), the contribution of superoxide (O2•-) to miniSOG-generated ROS remains unclear. We measured the light-dependent O2•- production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1O2 and establish that DHE is a suitable indicator to measure O2•- production in a system that produces both 1O2 and O2•-. We report that miniSOG produces both 1O2 and O2•-, as can its free chromophore, flavin mononucleotide. miniSOG produced O2•- at a rate of ~4.0µmol O2•-/min/µmol photosensitizer for an excitation fluence rate of 5.9mW/mm2 at 470 ± 20nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•- to miniSOG phenotypes should be considered.

Intermedin inhibits unilateral ureteral obstruction-induced oxidative stress via NADPH oxidase Nox4 and cAMP-dependent mechanisms.

NADPH oxidase Nox4-derived reactive oxygen species (ROS) play important roles in renal fibrosis. Our previous study demonstrated that intermedin (IMD) alleviated unilateral ureteral obstruction (UUO)-induced renal fibrosis by inhibition of ROS. However, the precise mechanisms remain unclear. Herein, we investigated the effect of IMD on Nox4 expression and NADPH oxidase activity in rat UUO model, and explored if these effect were achieved through cAMP-PKA pathway, the important post-receptor signal transduction pathway of IMD, in TGF-ß1-stimulated rat proximal tubular cell (NRK-52E). Renal fibrosis was induced by UUO. NRK-52E was exposed to rhTGF-ß1 to establish an in vitro model of fibrosis. IMD was overexpressed in the kidney and in NRK-52E by IMD gene transfer. We studied UUO-induced ROS by measuring dihydroethidium levels and lipid peroxidation end-product 4-hydroxynonenal expression. Nox4 expression in the obstructed kidney of UUO rat or in TGF-ß1-stimulated NRK-52E was measured by quantitative RT-PCR and Western blotting. We analyzed NADPH oxidase activity using a lucigenin-enhanced chemiluminescence system. We showed that UUO-stimulated ROS production was remarkably attenuated by IMD gene transfer. IMD overexpression inhibited UUO-induced up-regulation of Nox4 and activation of NADPH oxidase. Consistent with in vivo results, TGF-ß1-stimulated increase in Nox4 expression and NADPH oxidase activity was blocked by IMD. In NRK-52E, these beneficial effects of IMD were abolished by pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a PKA inhibitor, and mimicked by a cell-permeable cAMP analog dibutyl-cAMP. Our results indicate that IMD exerts anti-oxidant effects by inhibition of Nox4, and the effect can be mediated by cAMP-PKA pathway.

Elevated oxidative stress in the aortic media of patients with bicuspid aortic valve.

OBJECTIVE: Congenital bicuspid aortic valve (BAV) is distinctly associated with the development of ascending aortopathy in adulthood, portending risk of both ascending aortic aneurysm and dissection. Our previous work implicated deficiency in oxidative stress response as a mediator of the BAV-associated aortopathy. We hypothesize that reactive oxygen species generation invokes elevated local oxidative tissue damage in ascending aorta of patients with BAV. METHODS: Ascending aortic specimens were obtained from patients undergoing elective aortic replacement and/or aortic valve replacement and during heart transplant operations. Levels of superoxide anion were measured via high-pressure liquid chromatography-based detection of 2-hydroxyethidium in aortic specimens. Lipid peroxidation and enzymatic activity of superoxide dismutase and peroxidase were quantified in aortic specimens. RESULTS: Superoxide anion production was elevated in aortic specimens from patients with nonaneurysmal BAV (n = 59) compared with specimens from patients with the morphologically normal tricuspid aortic valve (TAV, n = 38). Total superoxide dismutase activity was similar among aortic specimens from patients with TAV versus BAV (n = 27 and 26, respectively), whereas peroxidase activity was increased in aortic specimens from patients with BAV compared with specimens from patients with TAV (n = 14 for both groups). Lipid peroxidation was elevated in aortic specimens from BAV patients compared with TAV patients (n = 14 and 11, respectively). CONCLUSIONS: Superoxide anion accumulation and increased lipid peroxidation demonstrate that, despite increased peroxidase activity, the ascending aortopathy of patients with BAV involves oxidative stress. In addition, the absence of increased superoxide dismutase activity in BAV specimens indicates a deficiency in antioxidant defense. This suggests that the characteristic smooth muscle cell loss observed in BAV aortopathy may be a consequence of superoxide-mediated cell damage.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.

Age increases reactive oxygen species production in macrophages and potentiates oxidative damage after spinal cord injury.

Age potentiates neurodegeneration and impairs recovery from spinal cord injury (SCI). Previously, we observed that age alters the balance of destructive (M1) and protective (M2) macrophages; however, the age-related pathophysiology in SCI is poorly understood. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) contributes to reactive oxygen species (ROS)-mediated damage and macrophage activation in neurotrauma. Further, NOX and ROS increase with central nervous system age. Here, we found significantly higher ROS generation in 14 versus 4-month-old (MO) mice after contusion SCI. Notably, NOX2 increased in 14 MO ROS-producing macrophages suggesting that macrophages and NOX contribute to SCI oxidative stress. Indicators of lipid peroxidation, a downstream cytotoxic effect of ROS accumulation, were significantly higher in 14 versus 4 MO SCI mice. We also detected a higher percentage of ROS-producing M2 (Arginase-1-positive) macrophages in 14 versus 4 MO mice, a previously unreported SCI phenotype, and increased M1 (CD16/32-positive) macrophages with age. Thus, NOX and ROS are age-related mediators of SCI pathophysiology and normally protective M2 macrophages may potentiate secondary injury through ROS generation in the aged injured spinal cord.

TEMPOL increases NAD(+) and improves redox imbalance in obese mice.

Continuous energy conversion is controlled by reduction-oxidation (redox) processes. NAD(+) and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD(+) production in the ascorbic acid-glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD(+)/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD(+)/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity.

Mechanical and Morphological Analysis of Cancer Cells on Nanostructured Substrates.

Cancer metastasis is a major cause of cancer-induced deaths in patients. Mimicking nanostructures of an extracellular matrix surrounding cancer cells can provide useful clues for metastasis. This paper compares the morphology, proliferation, spreading, and stiffness of highly aggressive glioblastoma multiforme cancer cells and normal fibroblast cells seeded on a variety of ordered polymeric nanostructures (nanopillars and nanochannels). Both cell lines survive and proliferate on the nanostructured surface and show more similarity on nanostructured surfaces than on flat surfaces. Although both show similar stiffness on the nanochannel surface, glioblastomas are softer, spread to a larger area, and elongate less than fibroblasts. The nanostructured surfaces are useful for in vitro model of an extracellular matrix to study the cancer cell migratory phenotype.

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification.

PURPOSE: To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs(1)). METHODS: Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1(+) hSSC(1) function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb(2)), ii) machined Ti surface group in osteogenic media (Mo(3)), iii) LASER-modified Ti group in basal media (Lb(4)) and, iv) LASER-modified Ti group in osteogenic media (Lo(5)). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP(6)) specific activity), live/dead immunostaining (Cell Tracker Green (CTG(7))/Ethidium Homodimer-1 (EH-1(8))), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype. RESULTS: Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs(1) on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP(6) specific activity on the hSSCs(1)-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP(6) and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated. CONCLUSIONS: LASER-modified Ti surfaces modify the behaviour of hSSCs.(1) In particular, SSC(1) adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were affected. The current studies show Ti LASER modification can enhance the osseointegration between Ti and skeletal cells, with important implications for orthopaedic application.