Hydroxyl Radical Antioxidant Capacity Research Articles

An in vitro analysis of an innovative standardized phospholipid carrier-based Melissa officinalis L. extract as a potential neuromodulator for emotional distress and related conditions.

Background: Melissa officinalis L. (MO), commonly known as lemon balm, a member of the mint family, is considered a calming herb. In various traditional medicines, it has been utilized to reduce stress and anxiety and promote sleep. A growing body of clinical evidence suggests that MO leaf extract supplementation possesses considerable neuropharmacological properties. However, its possible mechanism of action largely remains unknown. Objective: In the present in vitro studies, we comparatively investigated the central nervous system (CNS)-calming and antioxidative stress properties of an innovative standardized phospholipid carrier-based (Phytosome™) MO extract (Relissa™) vs. an unformulated dry MO extract. Methods: The neuropharmacological effect of the extract was studied in the anti-depressant enzymes γ-aminobutyrate transaminase (GABA-T) and monoamine oxidase A (MAO-A) assays and SH-SY5Y cells brain-derived neurotrophic factor (BDNF) expression assay. The neuroprotective effect of the extract against oxidative stress was assessed in SH-SY5Y cell-based (H2O2-exposed) Total Antioxidant Status (TAS) and Total Reactive Oxygen Species (ROS) assays. The cytotoxic effect of the extract was evaluated using MTT and LDH assays. The extract antioxidant effect was also evaluated in cell-free chemical tests, including TEAC-ABTS, DPPH, Ferric Reducing Antioxidant Power (FRAP), Oxygen Radical Antioxidant Capacity (ORAC), and Hydroxyl Radical Antioxidant Capacity (HORAC) assays. Results: Relissa™ exhibited high GABA-T inhibitory activity, IC50 (mg/mL) = 0.064 vs. unformulated dry MO extract, IC50 (mg/mL) = 0.27. Similar inhibitory effects were also observed for MAO-A. Relissa™ demonstrated an improved neuroprotective antioxidant effect on SH-SY5Y cells against H2O2-induced oxidative stress. Compared to unformulated dry MO extract, Relissa™ exerted high protective effect on H2O2-exposed SH-SY5Y cells, leading to higher cells BDNF expression levels. Moreover, cell-free chemical tests, including TEAC-ABTS, DPPH radical scavenging, FRAP, ORAC, and HORAC assays, validated the improved antioxidant effect of Relissa™ vs. unformulated dry MO extract. Conclusion: The results of the present study support the neuromodulating and neuroprotective properties of Relissa™, and its supplementation may help in the amelioration of emotional distress and related conditions.

Tuning oxidant and antioxidant activities of ceria by anchoring copper single-site for antibacterial application

The reaction system of hydrogen peroxide (H2O2) catalyzed by nanozyme has a broad prospect in antibacterial treatment. However, the complex catalytic activities of nanozymes lead to multiple pathways reacting in parallel, causing uncertain antibacterial results. New approach to effectively regulate the multiple catalytic activities of nanozyme is in urgent need. Herein, Cu single site is modified on nanoceria with various catalytic activities, such as peroxidase-like activity (POD) and hydroxyl radical antioxidant capacity (HORAC). Benefiting from the interaction between coordinated Cu and CeO2 substrate, POD is enhanced while HORAC is inhibited, which is further confirmed by density functional theory (DFT) calculations. Cu-CeO2 + H2O2 system shows good antibacterial properties both in vitro and in vivo. In this work, the strategy based on the interaction between coordinated metal and carrier provides a general clue for optimizing the complex activities of nanozymes.

Improved biocompatibility of dendrimer-based gene delivery by histidine-modified nuclear localization signals

Polyamidoamine (PAMAM) dendrimers have been explored as an alternative to polyethylenimine (PEI) as a gene delivery carrier because of their relatively low cytotoxicity and excellent biocompatibility. The transfection efficiency of PAMAM dendrimers can be improved by the addition of nuclear localization signal (NLS), a positively charged peptide sequence recognized by cargo proteins in the cytoplasm for nuclear transport. However, increased positive charges from NLS can cause damage to the cytoplasmic and mitochondrial membranes and lead to reactive oxygen species (ROS)-induced cytotoxicity. This negative effect of NLS can be negated without a significant reduction in transfection efficiency by adding histidine, an essential amino acid known as a natural antioxidant, to NLS. However, little is known about the exact mechanism by which histidine reduces cytotoxicity of NLS-modified dendrimers. In this study, we selected cystamine core PAMAM dendrimer generation 2 (cPG2) and conjugated it with NLS derived from Merkel cell polyomavirus large T antigen and histidine (n = 0–3) to improve transfection efficiency and reduce cytoxicity. NLS-modified cPG2 derivatives showed similar or higher transfection efficiency than PEI 25 kDa in NIH3T3 and human mesenchymal stem cells (hMSC). The cytotoxicity of NLS-modified cPG2 derivatives was substantially lower than PEI 25 kDa and was further reduced as the number of histidine in NLS increased. To understand the mechanism of cytoprotective effect of histidine-conjugated NLS, we examined ROS scavenging, hydroxyl radical generation and mitochondrial membrane potential as a function of the number of histidine in NLS. As the number of hisidine increased, cPG2 scavenged ROS more effectively as evidenced by the hydroxyl radical antioxidant capacity (HORAC) assay. This was consistent with the reduced intracellular hydroxyl radical concentration measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) assay in NIH3T3. Finally, fluorescence imaging with JC-1 confirmed that the mitochondrial membranes of NIH 3T3 were well-protected during the transfection when NLS contained histidine. These experimental results confirm the hypothesis that histidine residues scavenge ROS that is generated during the transfection process, preventing the excessive damage to mitochondrial membranes, leading to reduced cytotoxicity.

Chemical constituents and antioxidant capacities of Asparagus africanus Lam.

Phytochemical study of the roots and aerial parts of Asparagus africanus Lam. have led to the identification of thirty-three specialized metabolites ( 1–33 ) including two undescribed steroidal saponins: (25 S )− 5 β -spirostan-3 β -ol-3- O - β - d -xylopyranosyl-(1→2)- β - d -glucopyranoside ( 4 ) and (25 S )− 5 β -spirostan-3 β -ol-3- O - β - d -glucopyranosyl-(1→6)-[ β - d -glucopyranosyl-(1→2)-] β - d -glucopyranoside ( 5 ) and eight undescribed norlignans Aspafricanol A-E ( 6–10 ), Aspafricanene ( 11 ) and Aspafricanone A-B ( 12–13 ). Their structures were characterized by using a combination of 1D, and 2 D NMR spectroscopic and mass spectrometry (HR-ESI-MS) analyses, as well as by comparison with literature values. The antioxidant potential of the phenolic constituents was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical antioxidant capacity (HORAC) and cupric ion reducing antioxidant capacity (CUPRAC) methods. The results revealed higher antioxidant capacity for syringaresinol ( 21 ) in DPPH, HORAC and CUPRAC assays with IC 50 values of 15.9 ± 0.3, 86.0 ± 6.9 and 18.0 ± 1.7 µg/mL, respectively, compared to the positive controls (ascorbic acid, quercetin and Trolox). Additionally, compounds 7 , 9 and 28 showed similar or stronger activities than the positive controls in DPPH (IC 50 = 33.3 ± 5.7, 40.3 ± 0.6 and 46.7 ± 2.9 µg/mL, respectively), HORAC (IC 50 = 74.7 ± 4.6, 66.7 ± 5.8 and 99.3 ± 1.1 µg/mL, respectively), and CUPRAC assays (IC 50 = 128.7 ± 9.6, 123.3 ± 3.0 and 59.0 ± 1.7 µg/mL, respectively). • Thirty-three compounds were isolated from Asparagus africanus Lam. • Eight norlignans and two steroidal saponins are newly described. • Isolated phenolic constituents were evaluated for their antioxidant activities. • Four compounds showed good antioxidant activity similar to the standard used as control. • The greatest antioxidant activity was observed for syringaresnol.

In vivo study of dose-dependent antioxidant efficacy of functionalized core\u2013shell yttrium oxide nanoparticles

Herein, we assess the dose-dependent antioxidant efficacy of ultrafine spherical functionalized core–shell yttrium oxide nanoparticles (YNPs) with a mean size of 7–8 nm and modified with poly EGMP (ethylene glycol methacrylate phosphate) and N-Fluorescein Acrylamide. The antioxidant properties of these nanoparticles were investigated in three groups of Sprague–Dawley rats (10 per group) exposed to environmental stress daily for 1 week and one control group. Groups 2 and 3 were intravenously injected twice a week with YNPs at 0.3 and 0.5 mg at 2nd and 5th day of environmental stress exposure respectively. Different samples of blood and serum were collected from all experimental groups at end of the experiment to measure oxidative biomarkers such as total antioxidant capacity (TAC), hydroxyl radical antioxidant capacity (HORAC), oxygen radical antioxidant capacity (ORAC), malondialdehyde (MDA), and oxidants concentration as hydrogen peroxide (H2O2). The liver, brain, and spleen tissues were collected for fluorescence imaging and histopathological examination in addition to brain tissue examination by transmission electron microscope (TEM). Inductively coupled plasma-mass spectrometry (ICP-MS) was used to estimate YNPs translocation and concentration in tissues which is consecutively dependent on the dose of administration. Depending on all results, poly EGMP YNPs (poly EGMP yttrium oxide nanoparticles) can act as a potent direct antioxidant in a dose-dependent manner with good permeability through blood–brain barrier (BBB). Also, the neuroprotective effect of YNPs opening the door to a new therapeutic approach for modulating oxidative stress–related neural disorders.Highlights• The dose-dependent antioxidant efficacy of ultrafine spherical functionalized core–shell yttrium oxide nanoparticles (YNPs) with a mean size of 7–8 nm and modified with poly EGMP (ethylene glycol methacrylate phosphate) and N-Fluorescein Acrylamide was assessed.• The dose of administration directly affecting the brain, liver, and spleen tissues distribution, retention, and uptake of YNPs and direct correlation between the absorbed amount and higher dose administered.• YNPs can act as a potent direct antioxidant in a dose-dependent manner with good permeability through blood–brain barrier (BBB).Graphical abstract

Decoy Nanozymes Enable Multitarget Blockade of Proinflammatory Cascades for the Treatment of Multi-Drug-Resistant Bacterial Sepsis.

Sepsis is a life-threatening organ dysfunction characterized by severe systemic inflammatory response to infection. Effective treatment of bacterial sepsis remains a paramount clinical challenge, due to its astonishingly rapid progression and the prevalence of bacterial drug resistance. Here, we present a decoy nanozyme-enabled intervention strategy for multitarget blockade of proinflammatory cascades to treat multi-drug-resistant (MDR) bacterial sepsis. The decoy nanozymes (named MCeC@MΦ) consist mesoporous silica nanoparticle cores loaded with CeO2 nanocatalyst and Ce6 photosensitizer and biomimetic shells of macrophage membrane. By acting as macrophage decoys, MCeC@MΦ allow targeted photodynamic eradication of MDR bacteria and realize simultaneous endotoxin/proinflammatory cytokine neutralization. Meanwhile, MCeC@MΦ possess intriguing superoxide dismutase and catalase-like activities as well as hydroxyl radical antioxidant capacity and enable catalytic scavenging of multiple reactive oxygen species (ROS). These unique capabilities make MCeC@MΦ to collaboratively address the issues of bacterial infection, endotoxin/proinflammatory cytokine secretion, and ROS burst, fully cutting off the path of proinflammatory cascades to reverse the progression of bacterial sepsis. In vivo experiments demonstrate that MCeC@MΦ considerably attenuate systemic hyperinflammation and rapidly rescue organ damage within 1 day to confer higher survival rates (>75%) to mice with progressive MDR Escherichia coli bacteremia. The proposed decoy nanozyme-enabled multitarget collaborative intervention strategy offers a powerful modality for bacterial sepsis management and opens up possibilities for the treatment of cytokine storm in the COVID-19 pandemic and immune-mediated inflammation diseases.

Antioxidant and angiotensin I-converting enzyme (ACE) inhibitory peptides of rainbow trout (Oncorhynchus mykiss) viscera hydrolysates subjected to simulated gastrointestinal digestion and intestinal absorption

The objective of this work was to evaluate in vitro bioaccessibility, intestinal absorption, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of peptides from rainbow trout viscera hydrolysate (H). Rainbow trout Viscera (V) was hydrolyzed by Alcalase® 2.4L and a degree of hydrolysis (DH) of 44.8 ± 2.5% was achieved. Viscera and its hydrolysate were subjected to simulated gastrointestinal digestion (SGID) and intestinal absorption across Caco-2/TC7 cell monolayers. After the hydrolysis with Alcalase® 2.4L and the SGID of V, the species between 60.6 kDa and 13.0 kDa were decreased, causing an increase in species less than 6.51 kDa. The SGID of H did not modify the oxygen radical absorbance capacity (ORAC) or ACE inhibitory values but caused a significant decrease in the hydroxyl radical antioxidant capacity (HORAC) (30.2%). It also produced an increase in ABTS radical cation (ABTS assay) scavenging activity and ferric reducing antioxidant power (FRAP) (9.46% and 20.2%, respectively). Bioactive peptides in H were stable after SGID and they were partially able to cross Caco-2/TC7 cell monolayer, which demonstrates their possible intestinal absorption and their potential to act inside the organism.

Evolution of the anticholinesterase, antioxidant, and anti-inflammatory activity of Epilobium angustifolium L. infusion during in vitro digestion

• The aqueous infusion from fireweed exhibits potent anticholinesterase activity. • In vitro digestion influenced the evolution of anti-AChE activity. • Anti-AChE activity was correlated with total polyphenolic content in digestive fluids. • Antioxidant/reducing methods confirmed the activity of the fireweed infusion. Fireweed ( Epilobium angustifolium L.) has attracted attention due to its numerous biological activities. In the present work, we produced a polyphenol-rich freeze-dried infusion (407.02 ± 7.10 mg GAE/g) in order to measure, for the first time, the evolution of anticholinesterase activity during in vitro digestion. During the ‘gastric’ phase, there was an increase in the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity followed by a decrease during the ‘small intestinal’ phase. A strong correlation between total polyphenolic content (TPC) and the anticholinesterase activity of the digested fluids was observed (r = 0.90 for TPC/AChE; r = 0.88 for TPC/BChE, p < 0.05). Anti-cyclooxygenase-2 (COX-2) activity was significantly influenced by the digestion phase. Antioxidant/reducing assays (with ABTS and DPPH radicals, linoleic acid, or β-carotene; Cupric Reducing Antioxidant Capacity, Ferric Antioxidant Power, Hydroxyl Radical Antioxidant Capacity; and inhibition of superoxide dismutase, glutathione peroxidase, and glutathione reductase activity) yielded mixed results, but all of these methods confirmed activity during the digestion processes.

Analytical Methods Used in Determining Antioxidant Activity: A Review.

The study of antioxidants and their implications in various fields, from food engineering to medicine and pharmacy, is of major interest to the scientific community. The present paper is a critical presentation of the most important tests used to determine the antioxidant activity, detection mechanism, applicability, advantages and disadvantages of these methods. Out of the tests based on the transfer of a hydrogen atom, the following were presented: the Oxygen Radical Absorption Capacity (ORAC) test, the Hydroxyl Radical Antioxidant Capacity (HORAC) test, the Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) test, and the Total Oxyradical Scavenging Capacity (TOSC) test. The tests based on the transfer of one electron include the Cupric Reducing Antioxidant Power (CUPRAC) test, the Ferric Reducing Antioxidant Power (FRAP) test, the Folin–Ciocalteu test. Mixed tests, including the transfer of both a hydrogen atom and an electron, include the 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test, and the [2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl] (DPPH) test. All these assays are based on chemical reactions and assessing the kinetics or reaching the equilibrium state relies on spectrophotometry, presupposing the occurrence of characteristic colours or the discolouration of the solutions to be analysed, which are processes monitored by specific wavelength adsorption. These assays were successfully applied in antioxidant analysis or the determination of the antioxidant capacity of complex samples. As a complementary method in such studies, one may use methods based on electrochemical (bio)sensors, requiring stages of calibration and validation. The use of chemical methods together with electrochemical methods may result in clarification of the operating mechanisms and kinetics of the processes involving several antioxidants.

Isolation of Strong Antioxidants from Paeonia Officinalis Roots and Leaves and Evaluation of Their Bioactivities.

Paeonia officinalis extracts from leaves and roots were tested for their antioxidant potential using in vitro chemical (Folin-Ciocalteu, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), oxygen radical absorbance capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), hydroxyl radical scavenging capacity HOSC)) and cellular antioxidant activity (CAA) assays. Leaf extracts were stronger antioxidants than root extracts, while methanol was a more effective solvent than water in chemical assays. However, the selected water extract of leaves was a stronger antioxidant in CAA than the methanol extract (0.106 vs. 0.046 µmol quercetin equivalents/mg). Twenty compounds were identified by ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF) mass spectrometer, while on-line screening of their antioxidant capacity by high performance liquid chromatography (HPLC) with a DPPH•-scavenging detector revealed that gallic acid derivatives are the major peony antioxidants. Root water and leaf methanol extracts inhibited α-amylase in a dose dependent manner. The IC50 value for the strongest inhibitor, the methanol extract of leaves, was 1.67 mg/mL. In addition, the cytotoxicity assessment of extracts using human Caco-2 cells demonstrated that none of them possessed cytotoxic effects.

Changes in vascular and inflammatory biomarkers after exercise rehabilitation in patients with symptomatic peripheral artery disease

ObjectiveHome-based exercise is an alternative exercise mode to a structured supervised program to improve symptoms in patients with peripheral artery disease (PAD), but little is known about whether the slow-paced and less intense home program also elicits changes in vascular and inflammatory biomarkers. In an exploratory analysis from a randomized controlled trial, we compared changes in vascular and inflammatory biomarkers in patients with symptomatic PAD (typical and atypical of claudication) after home-based exercise and supervised exercise programs and in an attention-control group. MethodsA total of 114 patients were randomized into one of the three groups (n = 38 per group). Two groups performed exercise interventions, consisting of home-based and supervised programs of intermittent walking to mild to moderate claudication pain for 12 weeks; a third group performed light resistance training as a nonwalking attention-control group. Before and after intervention, patients were characterized on treadmill performance and endothelial effects of circulating factors present in sera by a cell culture-based bioassay on primary human arterial endothelial cells, and they were further evaluated on circulating vascular and inflammatory biomarkers. ResultsTreadmill peak walking time increased (P = .008) in the two exercise groups but not in the control group (P > .05). Cultured endothelial cell apoptosis decreased after home-based exercise (P < .001) and supervised exercise (P = .007), and the change in the exercise groups combined was different from that in the control group (P = .005). For circulating biomarkers, increases were found in hydroxyl radical antioxidant capacity (P = .003) and vascular endothelial growth factor A (P = .037), and decreases were observed in E-selectin (P = .007) and blood glucose concentration (P = .012) after home-based exercise only. The changes in hydroxyl radical antioxidant capacity (P = .005), vascular endothelial growth factor A (P = .008), and E-selectin (P = .034) in the exercise groups combined were different from those in the control group. ConclusionsThis exploratory analysis found that both home-based and supervised exercise programs are efficacious to decrease cultured endothelial cell apoptosis in patients with symptomatic PAD. Furthermore, a monitored home-based exercise program elicits additional vascular benefits by improving circulating markers of endogenous antioxidant capacity, angiogenesis, endothelium-derived inflammation, and blood glucose concentration in patients with symptomatic PAD. The novel clinical significance is that important trends were found in this exploratory analysis that a contemporary home-based exercise program and a traditional supervised exercise program may favorably improve vascular and inflammatory biomarkers in addition to the well-described ambulatory improvements in symptomatic patients with PAD.

Antioxidant capacity of soymilk yogurt and exopolysaccharides produced by lactic acid bacteria.

Reactive oxygen species (ROS), such as hydroxyl and superoxide anion radicals, are highly reactive molecules derived from the metabolism of oxygen. ROS play positive roles in cell physiology, but they may also damage cell membranes and DNA, inducing oxidation that causes membrane lipid peroxidation and decreases membrane fluidity. Soymilk yogurt, which is soymilk fermented using lactic acid bacteria (LAB), is an excellent food item with numerous functional substances with antioxidant effects. In this study, the antioxidative activities of soymilk yogurt were investigated. Sixteen of the 26 tested LAB strains solidified soymilk. In antioxidant capacity tests for bacterial cells, Leuconostoc mesenteroides MYU 60 and Pediococcus pentosaceus MYU 759 showed the highest values in the oxygen radical antioxidant capacity (ORAC) and hydroxyl radical antioxidant capacity (HORAC) tests, respectively. The supernatant of soymilk yogurt made with Lactobacillus gasseri MYU 1 showed the highest ORAC and HORAC values. L. mesenteroides MYU 60, Lactobacillus plantarum MYU 74, Lactobacillus reuteri MYU 220, and P. pentosaceus MYU 759 showed significantly high N-acetylcysteine equivalent values compared with the control in a total ROS reducing assay (p<0.05). These strains were selected, and a comet assay was performed, which exhibited decreased values in all selected strains compared with the control, indicating DNA protection. An acidic exopolysaccharide produced by P. pentosaceus MYU 759 showed high antioxidant capacity. The antioxidant substances produced by LAB fermentation may be exopolysaccharides, antioxidant peptides, and isoflavone aglycones. Soymilk yogurt can be used as a functional food useful for various diseases related to oxidation.

Peptide Inhibitor of Complement C1 (PIC1) demonstrates antioxidant activity via single electron transport (SET) and hydrogen atom transfer (HAT).

Reactive oxygen species (ROS) are natural byproducts of oxidative respiration that are toxic to organs and tissues. To mitigate ROS damage, organisms have evolved a variety of antioxidant systems to counteract these harmful molecules, however in certain pathological conditions these protective mechanisms can be overwhelmed. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1) mitigates peroxidase activity of the heme bearing proteins myeloperoxidase, hemoglobin, and myoglobin through a reversible process. To determine if this property of PIC1 was antioxidant in nature, we tested PIC1 in a number of well-established antioxidant assays. PIC1 showed dose-dependent antioxidant activity in a total antioxidant (TAC) assay, hydroxyl radical antioxidant capacity (HORAC) assay, oxygen radical antioxidant capacity (ORAC) assay as well as the thiobarbituric acid reactive substances (TBARS) assay to screen for PIC1 antioxidant activity in human plasma. The antioxidant activity of PIC1 in the TAC assay, as well as the HORAC/ORAC assay demonstrated that this peptide acts via the single electron transport (SET) and hydrogen atom transfer (HAT) mechanisms, respectively. Consistent with this mechanism of action, PIC1 did not show activity in a metal chelating activity (MCA) assay. PIC1 contains two vicinal cysteine residues and displayed similar antioxidant activity to the well characterized cysteine-containing tripeptide antioxidant molecule glutathione (GSH). Consistent with the role of the cysteine residues in the antioxidant activity of PIC1, oxidation of these residues significantly abrogated antioxidant activity. These results demonstrate that in addition to its described complement inhibiting activity, PIC1 displays in vitro antioxidant activity.

Abstract 20913: Endothelial Cell Inflammation and Antioxidant Capacity Are Associated With 6-Minute Walk Performance in Patients With Symptomatic Peripheral Artery Disease

Introduction: The aim of the study was to determine whether 6-minute walk total distance and pain-free distance were associated with circulating biomarkers of antioxidant capacity and inflammation, and with endothelial cell inflammation, oxidative stress, and apoptosis in patients with symptomatic peripheral artery disease (PAD). Methods: Exercise performance in 251 patients was assessed by pain-free distance and total distance covered during a 6-minute walk test. Patients were further characterized on clinical characteristics, on circulating biomarkers of antioxidant capacity and inflammation, and on the endothelial effects of circulating factors present in the sera using a cell culture-based bioassay on primary human arterial endothelial cells. Results: In a multivariate regression model, pain-free distance during the 6-minute walk test was negatively associated with dyslipidemia (p&lt;0.001), chronic kidney disease (p=0.004), and NF-κB (p=0.007), and was positively associated with height (p=0.008). In multivariate analyses, total distance walked during the 6-minute walk test was negatively associated with NF-κB (p&lt; 0.001), coronary artery disease (p=0.009), and BMI (p=0.022), and was positively associated with ABI (p&lt;0.001), male sex (p&lt;0.001), and hydroxyl radical antioxidant capacity (p&lt;0.001). Conclusions: 6-minute walk performance in symptomatic patients with PAD was associated with vascular biomarkers, as walking distances were negatively associated with endothelial cell inflammation and positively associated with circulating antioxidant capacity. The clinical implication is that behavioral interventions designed to alleviate endothelial cell inflammation and increase circulating antioxidant capacity, such as exercise and antioxidant intake, may improve ambulatory ability in patients with PAD during submaximal exercise that is typically performed during daily activities.

Endothelial Cell Inflammation and Antioxidant Capacity are Associated With 6-Minute Walk Performance in Patients With Symptomatic Peripheral Artery Disease.

We determined whether 6-minute walk total distance and pain-free distance were associated with circulating biomarkers of antioxidant capacity and inflammation and with cultured endothelial cell inflammation, oxidative stress, and apoptosis in 251 patients with symptomatic peripheral artery disease (PAD). In multivariate analyses, pain-free distance during the 6-minute walk test was negatively associated with dyslipidemia ( P < .001), chronic kidney disease ( P = .004), and transient transfection, nuclear factor κ-Light-Chain-Enhancer of activated B (NF-κB) cultured endothelial cells ( P = .007) and was positively associated with height ( P = .008). Furthermore, total distance walked during the 6-minute walk test was negatively associated with cultured endothelial cell NF-κB ( P < .001), coronary artery disease ( P = .009), and body mass index ( P = .022) and was positively associated with ankle-brachial index ( P < .001), male sex ( P < .001), and hydroxyl radical antioxidant capacity ( P < .001). The 6-minute walk performance in symptomatic patients with PAD was associated with vascular biomarkers, as walking distances were negatively associated with cultured endothelial cell inflammation and positively associated with circulating antioxidant capacity. The clinical implication is that behavioral interventions designed to alleviate endothelial cell inflammation and increase circulating antioxidant capacity, such as exercise and antioxidant intake, may improve ambulation of patients with PAD during submaximal exercise that is typically performed during daily activities.

Association between daily walking and antioxidant capacity in patients with symptomatic peripheral artery disease

ObjectiveThe primary aim of the study was to assess whether both the amount and pace of daily walking were associated with circulating antioxidant capacity in symptomatic patients with peripheral artery disease (PAD). MethodsCommunity-based walking was measured in 244 men and women who were limited by symptomatic PAD during a 1-week period in which they wore an ankle-mounted step activity monitor. Patients were further characterized by circulating antioxidant capacity with the OxiSelect (Cell Biolabs Inc, San Diego, Calif) hydroxyl radical antioxidant capacity (HORAC) activity assay. ResultsTo assess the amount of walking, patients were grouped into low (≤2440 strides/d), middle (2441-3835 strides/d), and high (>3835 strides/d) stride tertiles. HORAC was higher in the middle (P = .03) and high (P = .01) stride tertiles than in the low tertile, but there was no difference between middle and high tertiles (P = .44). To assess the pace of walking, patients were grouped into slow (<25.0 strides/min), middle (25.0-31.6 strides/min), and fast (>31.6 strides/min) cadence tertiles. HORAC was higher in the high cadence tertile than in the low (P < .01) and middle (P < .01) tertiles, but there was no difference between low and middle tertiles (P = .48). Similar findings were obtained on group differences in HORAC after adjusting for age, sex, race, and ankle-brachial index for both the amount and pace of daily walking. ConclusionsWalking >2440 strides each day and walking at a cadence faster than 31.6 strides/min for 30 minutes each day are both associated with greater circulating antioxidant capacity in symptomatic patients with PAD. The clinical significance is that a home-based walking program may be one approach to increase endogenous antioxidant capacity.

Abstract 12275: Knock-down of Insulin-like Growth Factor-1 Receptor in the Endothelium Decreases Anti-oxidant Capacity of Aorta, Leading to Increased Atherosclerosis

Systemic infusion of insulin-like growth factor-1 (IGF-1) in Apoe-deficient mice exerts anti-atherogenic effects by reducing oxidative stress and pro-inflammatory responses in lesions, yet precise mechanisms remain elusive. We have shown that smooth muscle specific IGF-1 overexpression does not reduce atherosclerotic burden, suggesting that smooth muscle cells do not mediate anti-atherogenic effects of IGF-1. The endothelium is integral in atherogenesis, however a potential role of endothelial IGF-1 receptor (IGF1R) signaling in atherogenesis is unknown. To investigate, we generated Apoe-deficient/endothelial IGF1R-knockdown (En-IGF1R-KD) mice, which are Igf1r-floxed and express Cre recombinase driven by the Cdh5-promoter. IGF1R expression levels were lower by 60% in endothelial cells isolated from En-IGF1R-KD mice compared to IGF1R-flox cells. After 12 weeks of high-fat diet feeding, En-IGF1R-KD mice had more atherosclerotic plaque than IGF1R-flox control (Oil Red O staining of en face aorta, 33±8% increase, P&lt;0.05), indicating that loss of endothelial IGF1R signaling enhanced atherogenesis. Intriguingly, En-IGF1R-KD did not alter systolic blood pressure (IGF1R-flox; 119.5±2.6 vs. En-IGF1R-KD; 119.1±2.0 mmHg) or plasma nitrate/nitrite levels (index of nitric oxide bioavailability, IGF1R-flox; 8.6±0.4 vs. En-IGF1R-KD; 7.3±0.9 uM). Vasorelaxation to acetylcholine (Ach) was equivalent in aortas of En-IGF1R-KD and IGF1R-flox mice after high-fat diet feeding, suggesting that endothelial function was not affected by En-IGF1R-KD. Intriguingly, high-fat diet feeding made aortas hyporesponsive to Ach both in En-IGF1R-KD and IGF1R-flox mice, and in normal chow fed mice En-IGF1R-KD aortas were more responsive to Ach than IGF1R-flox aortas, indicating that loss of IGF-1 signaling improved endothelial function. On the other hand, anti-oxidant capacity as measured by Hydroxyl Radical Antioxidant Capacity Activity Assay was significantly lower in En-IGF1R-KD aorta by 30.0±6.7%, consistent with anti-oxidant effects of IGF-1 on endothelial cells. In summary, our results indicate that IGF1R signaling in the endothelium is atheroprotective, and that this atheroprotective effect is mediated via an anti-oxidant mechanism.

Endothelial Cell Inflammation and Antioxidant Capacity are Associated With Exercise Performance and Microcirculation in Patients With Symptomatic Peripheral Artery Disease.

We determined whether exercise performance and lower extremity microcirculation were associated with endothelial cell inflammation, oxidative stress, and apoptosis and with circulating biomarkers of inflammation and antioxidant capacity in 160 patients with symptomatic peripheral artery disease (PAD). In a multivariate regression model for peak walking time, significant independent variables included ankle-brachial index (P < .001), age (P = .017), hydroxyl radical antioxidant capacity (P = .008), and endothelial cell nuclear factor K-light-chain-enhancer of activated B cells (NF-κB) activity (P = .015). In multivariate analyses for time to minimum exercise calf muscle hemoglobin oxygen saturation (StO2), significant independent variables included endothelial cell NF-κB activity (P = .043) and calf muscle StO2 at rest (P = .007). Endothelial cell inflammation and circulating biomarkers of inflammation and antioxidant capacity were associated with exercise performance and microcirculation of the ischemic calf musculature during exercise. The clinical implication is that interventions designed to alleviate endothelial cell inflammation and circulating inflammatory biomarkers, such as antioxidant therapy, may improve exercise performance of symptomatic patients with PAD.

Abstract 12280: Endothelial Cell Inflammation and Antioxidant Capacity are Associated With Exercise Performance and Microcirculation in Patients With Peripheral Artery Disease

Background: We determined whether exercise performance and lower extremity microcirculation were associated with endothelial cell inflammation, oxidative stress, and apoptosis, and with circulating biomarkers of inflammation and antioxidant capacity. Methods: One hundred sixty symptomatic patients with PAD were characterized on the endothelial effects of circulating factors present in the sera using a cell culture-based bioassay on primary human arterial endothelial cells. Patients were further evaluated on circulating inflammatory and vascular biomarkers, physical examination, medical history, exercise performance measured during treadmill evaluation by peak walking time (PWT), and claudication onset time (COT), and lower extremity microcirculation measured by calf muscle hemoglobin oxygen saturation (StO 2 ) during treadmill exercise. Results: In the multivariate regression model for PWT, the significant independent variables included ankle-brachial index (p&lt;0.001), age (p=0.017), hydroxyl radical antioxidant capacity (HORAC) (p=0.008), and endothelial cell NF-κB activity (p=0.015). In the multivariate regression model for COT, the significant independent variable was endothelial cell NF-κB activity (p=0.013). In the multivariate analyses for the average rate of decline in calf muscle StO 2 during exercise, the significant independent variables included body mass index (p&lt;0.001) and HORAC (p=0.024). Conclusions: Endothelial cell inflammation and circulating biomarkers of inflammation and antioxidant capacity are significant factors for exercise performance and microcirculation of the ischemic calf musculature during exercise. The clinical implication is that interventions designed to alleviate endothelial cell inflammation and circulating inflammatory biomarkers, such as antioxidant therapy, may improve exercise performance of symptomatic patients with PAD.

Greater endothelial apoptosis and oxidative stress in patients with peripheral artery disease.

We compared apoptosis, cellular oxidative stress, and inflammation of cultured endothelial cells treated with sera from 156 subjects with peripheral artery disease (PAD) and 16 healthy control subjects. Furthermore, we compared circulating inflammatory, antioxidant capacity, and vascular biomarkers between the two groups. The PAD group had a 164% higher value for endothelial cell apoptosis (P < 0.001) and a 62% higher value for endothelial cellular reactive oxygen species production (P < 0.001) than the control group. Furthermore, the PAD group had lower systemic antioxidant capacity measured by hydroxyl radical antioxidant capacity activity (P < 0.001), higher inflammatory and vascular measures of high-sensitivity C-reactive protein (P < 0.001), interleukin-8 (P < 0.001), serum amyloid A (P < 0.001), vascular cell adhesion molecule-1 (P < 0.001), adiponectin (P < 0.001), apolipoprotein B (P = 0.013), apolipoprotein CIII (P = 0.035), lower vascular endothelial growth factor-A (P < 0.001), and hepatocyte growth factor (P < 0.001) than the control group. Subjects with PAD have greater endothelial apoptosis and oxidative stress than control subjects with low burden of comorbid conditions and cardiovascular risk factors. Furthermore, subjects with PAD have lower systemic antioxidant capacity and angiogenic measures and higher circulating inflammatory parameters.