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Acute-phase serum amyloid A (SAA) can disrupt vascular homeostasis and is elevated in subjects with diabetes, cardiovascular disease, and rheumatoid arthritis. Cyclic nitroxides (e.g., Tempo) are a class of piperidines that inhibit oxidative stress and inflammation. This study examined whether 4-methoxy-Tempo (4-MetT) inhibits SAA-mediated vascular and renal dysfunction. Acetylcholine-mediated vascular relaxation and aortic guanosine-3′,5′-cyclic monophosphate (cGMP) levels both diminished in the presence of SAA. 4-MetT dose-dependently restored vascular function with corresponding increases in cGMP. Next, male ApoE-deficient mice were administered a vehicle (control, 100 µL PBS) or recombinant SAA (100 µL, 120 µg/mL) ± 4-MetT (at 15 mg/kg body weight via i.p. injection) with the nitroxide administered before (prophylaxis) or after (therapeutic) SAA. Kidney and hearts were harvested at 4 or 16 weeks post SAA administration. Renal inflammation increased 4 weeks after SAA treatment, as judged by the upregulation of IFN-γ and concomitant increases in iNOS, p38MAPK, and matrix metalloproteinase (MMP) activities and increased renal fibrosis (Picrosirius red staining) in the same kidneys. Aortic root lesions assessed at 16 weeks revealed that SAA enhanced lesion size (vs. control; p < 0.05), with plaque presenting with a diffuse fibrous cap (compared to the corresponding aortic root from control and 4-MetT groups). The extent of renal dysfunction and aortic lesion size was largely unchanged in 4-MetT-supplemented mice, although renal fibrosis diminished at 16 weeks, and aortic lesions presented with redistributed collagen networks. These outcomes indicate that SAA stimulates renal dysfunction through promoting the IFN-γ–iNOS–p38MAPK axis, manifesting as renal damage and enhanced atherosclerotic lesions, while supplementation with 4-MetT only affected some of these pathological changes.

IntroductionCrohn’s disease (CD) involves activation of mast cells (MC) and NF-кB in parallel with the PPAR-α/NLRP3 inflammasome/IL-1β pathway in the inflamed colon. Whether polyphenols from maqui (Aristotelia chilensis) represent a natural alternative treatment for CD is unclear. Therefore, we used an animal model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD-like colitis to investigate protective effects of maqui extract through monitoring NLRP3 inflammasome and MC activation in colon tissue.MethodsMaqui extract was administered via orogastric route to mice after (post-Treatment group) or prior (pre-Treatment group) to TNBS-induction. Colon pathology was characterized by histoarchitectural imaging, disease activity index (DAI), and assessing NF-кB, p-NF-кB, PPAR-α/NLRP3 expression and IL-1β levels.ResultsCompared to mice treated with TNBS alone administration of anthocyanin-rich maqui extract improved the DAI, colon histoarchitecture and reduced both colon wet-weight and transmural inflammation. Induction with TNBS significantly increased colonic NLPR3 inflammasome activation, while co-treatment with maqui extract (either post- or pre-Treatment) significantly downregulated NLRP3, ASC and caspase-1 levels, which manifested as reduced colonic IL-1β levels. Supplemented maqui extract marginally diminished NF-кB activity in epithelial cells but reached statistical significance in immune cells (as judged by decreased NF-кB phosphorylation). PPAR-α signaling was largely unaffected by Maqui whereas MC infiltration into the colon mucosa and submucosa decreased and their level of degranulation was suppressed.ConclusionThese outcomes show the post- and pre- Treatment effect of a polyphenolic extract rich in anthocyanins from maqui the acute phase of TNBS- induced CD-like colitis is linked to suppression of the NLRP3 inflammasome and reduced MC responses. These data indicate that maqui extract represents a potential nutraceutical for the treatment of inflammatory bowel disease (IBD).

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

Abstract We investigated the association of dietary intake of major types of fatty acids with heart disease mortality in a general adult cohort with or without a prior diagnosis of myocardial infarction (MI). This cohort study included US adults who attended the National Health and Nutrition Examination Surveys from 1988 to 2014. Heart disease mortality was ascertained by linkage to the National Death Index records through 31 December 2015. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of fatty acid intake for heart disease mortality. This cohort included 45,820 adults among which 1,541 had a prior diagnosis of MI. Participants were followed up for 532,722 person-years (mean follow-up, 11.6 years), with 2,313 deaths recorded from heart disease being recorded. Intake of saturated (SFAs) and monounsaturated fatty acids (MUFAs) was associated with heart disease mortality after adjustment for all the tested confounders. In contrast, a 5% higher calorie intake from polyunsaturated fatty acids (PUFAs) was associated with a 9% (HR, 0.91; 95% CI 0.83–1.00; P = 0.048) lower multivariate-adjusted risk of heart disease mortality. Sub-analyses showed that this inverse association was present in those without a prior diagnosis of MI (HR,0.89; 95% CI 0.80–0.99) but not in those with the condition (HR, 0.94; 95% CI 0.75–1.16). The lack of association in the MI group could be due to a small sample size or severity and procedural complications (e.g., stenting and medication adherence) of the disease. Higher PUFA intake was associated with a favourable lipid profile. However, further adjustment for plasma lipids did not materially change the inverse association between PUFAs and heart disease mortality. Higher intake of PUFAs, but not SFAs and MUFAs, was associated with a lower adjusted risk of heart disease mortality in a large population of US adults supporting the need to increase dietary PUFA intake in the general public.

Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.

Abstract The treatment of periodontitis has numerous positive effects on established chronic health conditions, including cardiovascular disease and diabetes. However, ethical considerations do limit the establishment of human trials to investigate whether periodontitis promotes the early stages of chronic conditions. Therefore, the aim of this study was to investigate whether periodontitis induces endothelial dysfunction in hyperlipidemic apolipoprotein E gene-deficient (ApoE-/-) mice. Forty-five 8-week-old ApoE-/- mice were challenged by oral lavage with Porphyromonas gingivalis and Streptococcus gordonii for 4 weeks. A subgroup of animals (n = 15–17/group) was placed in a metabolic chamber immediately before euthanasia at 4 weeks to measure VO2/CO2 concentrations and voluntary locomotion. In infected and control animals alveolar bone levels were measured by x-ray imaging and endothelial function was determined by measuring endothelial-dependent vasorelaxation of aortic rings. The mRNA expression levels of serum amyloid A and tumor necrosis factor were determined in liver tissues by qRT PCR and protein concentrations in serum by ELISA. Caecal contents were analysed by sequencing to determine changes to the gut microbiota to investigate linkages between microbiome and systemic changes. The results showed that oral lavage of P. gingivalis and S. gordonii for 4 weeks, initiated periodontitis in ApoE-/- mice, similar to the human situation. The oral inflammation was accompanied by a significant increase in mRNA expression of pro-inflammatory mediators serum amyloid A1 and tumor necrosis factor in the liver. Mice with periodontitis also exhibited impaired endothelial-dependent vasorelaxation responses to acetylcholine. This systemic response was connected to increased energy expenditure, locomotion and respiratory quotient. No differences were detected in caecal microbiota between the infected and control animals. Overall, this is the first report that provide evidence that periodontitis induces endothelial dysfunction in mice. Other systemic responses observed in response to the local reaction need further investigation. The study suggests that early prevention of periodontitis may help limit the early stages of endothelial dysfunction that is linked to atherogenesis in humans.

Innate and adaptive immune responses comprise a complex network of protein–protein and protein–cell interactions that regulates commensal flora and protects organisms from foreign pathogens and transformed (proliferating) host cells under physiological conditions such as pregnancy, growth and development as well as formulating a response pathological challenge [...]

Dietary selenium (Se) intake within the physiological range is critical to maintain various biological functions, including antioxidant defence, redox homeostasis, growth, reproduction, immunity, and thyroid hormone production. Chemical forms of dietary Se are diverse, including organic Se (selenomethionine, selenocysteine, and selenium-methyl-selenocysteine) and inorganic Se (selenate and selenite). Previous studies have largely investigated and compared the health impacts of dietary Se on agricultural stock and humans, where dietary Se has shown various benefits, including enhanced growth performance, immune functions, and nutritional quality of meats, with reduced oxidative stress and inflammation, and finally enhanced thyroid health and fertility in humans. The emergence of nanoparticles presents a novel and innovative technology. Notably, Se in the form of nanoparticles (SeNPs) has lower toxicity, higher bioavailability, lower excretion in animals, and is linked to more powerful and superior biological activities (at a comparable Se dose) than traditional chemical forms of dietary Se. As a result, the development of tailored SeNPs for their use in intensive agriculture and as candidate for therapeutic drugs for human pathologies is now being actively explored. This review highlights the biological impacts of SeNPs on growth and reproductive performances, their role in modulating heat and oxidative stress and inflammation and the varying modes of synthesis of SeNPs.

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE−/−) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE−/− mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE−/− mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that arises due to a complex and variable interplay between elements including age, genetic, and environmental risk factors that manifest as the loss of dopaminergic neurons. Contemporary treatments for PD do not prevent or reverse the extent of neurodegeneration that is characteristic of this disorder and accordingly, there is a strong need to develop new approaches which address the underlying disease process and provide benefit to patients with this debilitating disorder. Mitochondrial dysfunction, oxidative damage, and inflammation have been implicated as pathophysiological mechanisms underlying the selective loss of dopaminergic neurons seen in PD. However, results of studies aiming to inhibit these pathways have shown variable success, and outcomes from large-scale clinical trials are not available or report varying success for the interventions studied. Overall, the available data suggest that further development and testing of novel therapies are required to identify new potential therapies for combating PD. Herein, this review reports on the most recent development of antioxidant and anti-inflammatory approaches that have shown positive benefit in cell and animal models of disease with a focus on supplementation with natural product therapies and selected synthetic drugs.

The cyclic nitroxide TEMPOL exerts anti-oxidative and anti-inflammatory effects, and thus may provide therapeutic benefit in Parkinson’s disease (PD), in which mitochondrial dysfunction, oxidative damage and inflammation have been implicated as pathophysiological mechanisms underlying the selective loss of dopaminergic neurons. Markers of oxidative stress and inflammation were investigated in a cell model of differentiated human neuroblastoma (SH-SY5Y) cells treated with the neurotoxin, 6-hydroxydopamine (6-OHDA). Treatment with TEMPOL ameliorated 6-OHDA-mediated cytotoxicity and attenuated biomarkers of oxidative stress including: mitochondrial superoxide anion free radical production, lipid peroxidation, induction of heme oxygenase 1 (HO-1) protein expression and NFκB activation. Treatment with TEMPOL abated decreased gene expression of DRD2S and DRD2L induced by 6-OHDA indicating that TEMPOL may prevent mitochondrial dysfunction and activation of pathways that result in receptor desensitization. 6-OHDA insult decreased gene expression of the antioxidant, SOD-1, and this diminution was also mitigated by TEMPOL. Activation of NFκB increased pro-inflammatory IFNy and decreased IL-6, however, TEMPOL had no effect on these inflammation mediators. Overall, this data suggests that cyclic nitroxides may preserve dopaminergic neuronal cell viability by attenuating oxidative stress and mitochondrial dysfunction, but are unable to affect inflammatory mediators that propagate cellular damage and neurodegeneration in PD.

Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p

Inflammatory bowel disease (IBD) is a chronic condition characterised by leukocyte recruitment to the gut mucosa. Leukocyte myeloperoxidase (MPO) produces the two-electron oxidant hypochlorous acid (HOCl), damaging tissue and playing a role in cellular recruitment, thereby exacerbating gut injury. We tested whether the MPO-inhibitor, 4-Methoxy-TEMPO (MetT), ameliorates experimental IBD. Colitis was induced in C57BL/6 mice by 3% w/v dextran-sodium-sulfate (DSS) in drinking water ad libitum over 9-days with MetT (15 mg/kg; via i. p. injection) or vehicle control (10% v/v DMSO+90% v/v phosphate buffered saline) administered twice daily during DSS challenge. MetT attenuated body-weight loss (50%, p

Acute serum amyloid A (SAA) is an apolipoprotein that mediates pro-inflammatory and pro-atherogenic pathways. SAA-mediated signalling is diverse and includes canonical and acute immunoregulatory pathways in a range of cell types and organs. This study aimed to further elucidate the roles for SAA in the pathogenesis of vascular and renal dysfunction. Two groups of male ApoE-deficient mice were administered SAA (100 µL, 120 µg/mL) or vehicle control (100 µL PBS) and monitored for 4 or 16 weeks after SAA treatment; tissue was harvested for biochemical and histological analyses at each time point. Under these conditions, SAA administration induced crosstalk between NF-κB and Nrf2 transcriptional factors, leading to downstream induction of pro-inflammatory mediators and antioxidant response elements 4 weeks after SAA administration, respectively. SAA treatment stimulated an upregulation of renal IFN-γ with a concomitant increase in renal levels of p38 MAPK and matrix metalloproteinase (MMP) activities, which is linked to tissue fibrosis. In the kidney of SAA-treated mice, the immunolocalisation of inducible nitric oxide synthase (iNOS) was markedly increased, and this was localised to the parietal epithelial cells lining Bowman’s space within glomeruli, which led to progressive renal fibrosis. Assessment of aortic root lesion at the study endpoint revealed accelerated atherosclerosis formation; animals treated with SAA also showed evidence of a thinned fibrous cap as judged by diffuse collagen staining. Together, this suggests that SAA elicits early renal dysfunction through promoting the IFN-γ-iNOS-p38 MAPK axis that manifests as the fibrosis of renal tissue and enhanced cardiovascular disease.

Intracellular redox imbalance in endothelial cells (EC) can lead to endothelial dysfunction, which underpins cardiovascular diseases (CVD). The acute phase serum amyloid A (SAA) elicits inflammation through stimulating production of reactive oxygen species (ROS). The cyclic nitroxide 4-MethoxyTEMPO (4-MetT) is a superoxide dismutase mimetic that suppresses oxidant formation and inflammation. The aim of this study was to investigate whether 4-MetT inhibits SAA-mediated activation of cultured primary human aortic EC (HAEC). Co-incubating cells with 4-MetT inhibited SAA-mediated increases in adhesion molecules (VCAM-1, ICAM-1, E-selectin, and JAM-C). Pre-treatment of cells with 4-MetT mitigated SAA-mediated increases in transcriptionally activated NF-κB-p65 and P120 Catenin (a stabilizer of Cadherin expression). Mitochondrial respiration and ROS generation (mtROS) were adversely affected by SAA with decreased respiratory reserve capacity, elevated maximal respiration and proton leakage all characteristic of SAA-treated HAEC. This altered respiration manifested as a loss of mitochondrial membrane potential (confirmed by a decrease in TMRM fluorescence), and increased mtROS production as assessed with MitoSox Red. These SAA-linked impacts on mitochondria were mitigated by 4-MetT resulting in restoration of HAEC nitric oxide bioavailability as confirmed by assessing cyclic guanosine monophosphate (cGMP) levels. Thus, 4-MetT ameliorates SAA-mediated endothelial dysfunction through normalising EC redox homeostasis. Subject to further validation in in vivo settings; these outcomes suggest its potential as a therapeutic in the setting of cardiovascular pathologies where elevated SAA and endothelial dysfunction is linked to enhanced CVD.

Elevated serum amyloid A (SAA) levels may promote endothelial dysfunction, which is linked to cardiovascular and renal pathologies. We investigated the effect of SAA on vascular and renal function in apolipoprotein E-deficient (ApoE−/−) mice. Male ApoE−/− mice received vehicle (control), low-level lipopolysaccharide (LPS), or recombinant human SAA by i.p. injection every third day for 2 weeks. Heart, aorta and kidney were harvested between 3 days and 18 weeks after treatment. SAA administration increased vascular cell adhesion molecule (VCAM)-1 expression and circulating monocyte chemotactic protein (MCP)-1 and decreased aortic cyclic guanosine monophosphate (cGMP), consistent with SAA inhibiting nitric oxide bioactivity. In addition, binding of labeled leukocytes to excised aorta increased as monitored using an ex vivo leukocyte adhesion assay. Renal injury was evident 4 weeks after commencement of SAA treatment, manifesting as increased plasma urea, urinary protein, oxidized lipids, urinary kidney injury molecule (KIM)-1 and multiple cytokines and chemokines in kidney tissue, relative to controls. Phosphorylation of nuclear-factor-kappa-beta (NFκB-p-P65), tissue factor (TF), and macrophage recruitment increased in kidneys from ApoE−/− mice 4 weeks after SAA treatment, confirming that SAA elicited a pro-inflammatory and pro-thrombotic phenotype. These data indicate that SAA impairs endothelial and renal function in ApoE−/− mice in the absence of a high-fat diet.

Reperfusion therapy increases survival post-acute myocardial infarction (AMI) while also stimulating secondary oxidant production and immune cell infiltration. Neutrophils accumulate within infarcted myocardium within 24 h post-AMI and release myeloperoxidase (MPO) that catalyses hypochlorous acid (HOCl) production while increasing oxidative stress and inflammation, thereby enhancing ventricular remodelling. Nitroxides inhibit MPO-mediated HOCl production, potentially ameliorating neutrophil-mediated damage. Aim: Assess the cardioprotective ability of nitroxide 4-methoxyTEMPO (4MetT) within the setting of AMI. Methods: Male Wistar rats were separated into 3 groups: SHAM, AMI/R, and AMI/R + 4MetT (15 mg/kg at surgery via oral gavage) and subjected to left descending coronary artery ligation for 30 min to generate an AMI, followed by reperfusion. One cohort of rats were sacrificed at 24 h post-reperfusion and another 28 days post-surgery (with 4MetT (15 mg/kg) administration twice daily). Results: 3-chlorotyrosine, a HOCl-specific damage marker, decreased within the heart of animals in the AMI/R + 4-MetT group 24 h post-AMI, indicating the drug inhibited MPO activity; however, there was no evident difference in either infarct size or myocardial scar size between the groups. Concurrently, MPO, NfκB, TNFα, and the oxidation marker malondialdehyde increased within the hearts, with 4-MetT only demonstrating a trend in decreasing MPO and TNF levels. Notably, 4MetT provided a significant improvement in cardiac function 28 days post-AMI, as assessed by echocardiography, indicating potential for 4-MetT as a treatment option, although the precise mechanism of action of the compound remains unclear.

Serum amyloid A (SAA) promotes endothelial inflammation and dysfunction that is associated with cardiovascular disease and renal pathologies. SAA is an apoprotein for high-density lipoprotein (HDL) and its sequestration to HDL diminishes SAA bioactivity. Herein we investigated the effect of co-supplementing HDL on SAA-mediated changes to vascular and renal function in apolipoprotein E-deficient (ApoE−/−) mice in the absence of a high-fat diet. Male ApoE−/− mice received recombinant human SAA or vehicle (control) by intraperitoneal (i.p.) injection every three days for two weeks with or without freshly isolated human HDL supplemented by intravenous (i.v.) injection in the two weeks preceding SAA stimulation. Aorta and kidney were harvested 4 or 18 weeks after commencement of treatment. At 4 weeks after commencement of treatment, SAA increased aortic vascular cell adhesion molecule (VCAM)-1 expression and F2-isoprostane level and decreased cyclic guanosine monophosphate (cGMP), consistent with SAA stimulating endothelial dysfunction and promoting atherosclerosis. SAA also stimulated renal injury and inflammation that manifested as increased urinary protein, kidney injury molecule (KIM)-1, and renal tissue cytokine/chemokine levels as well as increased protein tyrosine chlorination and P38 MAPkinase activation and decreased in Bowman’s space, confirming that SAA elicited a pro-inflammatory phenotype in the kidney. At 18 weeks, vascular lesions increased significantly in the cohort of ApoE−/− mice treated with SAA alone. By contrast, pretreatment of mice with HDL decreased SAA pro-inflammatory activity, inhibited SAA enhancement of aortic lesion size and renal function, and prevented changes to glomerular Bowman’s space. Taken together, these data indicate that supplemented HDL reduces SAA-mediated endothelial and renal dysfunction in an atherosclerosis-prone mouse model.

Pyocyanin secreted by Pseudomonas aeruginosa is a virulence factor that damages epithelial cells during infection through the action of reactive oxygen species, however, little is known about its direct effect on biofilms. We demonstrated that pyocyanin-producing P. aeruginosa strains (PA14WT, DKN370, AES-1R, and AES-2) formed robust biofilms in contrast to the poorly formed biofilms of the pyocyanin mutant PA14ΔphzA-G and the low pyocyanin producer AES-1M. Addition of DNase I and reduced glutathione (GSH) significantly reduced biofilm biomass of pyocyanin-producing strains (P < 0.05) compared to non-pyocyanin producers. Subsequently we showed that a combined treatment comprising: GSH + DNase I + antibiotic, disrupted and reduced biofilm biomass up to 90% in cystic fibrosis isolates AES-1R, AES-2, LESB58, and LES431 and promoted lung epithelial cell (A549) recovery and growth. We also showed that exogenously added GSH restored A549 epithelial cell glutathione reductase activity in the presence of pyocyanin through recycling of GSSG to GSH and consequently increased total intracellular GSH levels, inhibiting oxidative stress, and facilitating cell growth and confluence. These outcomes indicate that GSH has multiple roles in facilitating a return to normal epithelial cell growth after insult by pyocyanin. With increased antibiotic resistance in many bacterial species, there is an urgency to establish novel antimicrobial agents. GSH is able to rapidly and comprehensively destroy P. aeruginosa associated biofilms while at a same time assisting in the recovery of host cells and re-growth of damaged tissue.

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction—a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Exposure of the oxygen transport protein horse heart myoglobin (hhMb) to HOCl inhibits Iron III (Fe(III))-heme reduction by cytochrome b5 to oxygen-binding Iron II (Fe(II))Mb. Pathological concentrations of HOCl yielded myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/Vis absorbance. Mass analysis indicated HOCl caused successive mass increases of 16 a.m.u., consistent serial addition of molecular oxygen to the protein. By contrast, parallel analysis of protein chlorination by quantitative mass spectrometry revealed a comparatively minor increase in the 3-chlorotyrosine/tyrosine ratio. Pre-treatment of hhMb with HOCl affected the peroxidase reaction between the hemoprotein and H2O2 as judged by a HOCl dose-dependent decrease in spin-trapped tyrosyl radical detected by electron paramagnetic resonance (EPR) spectroscopy and the rate constant of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) oxidation. By contrast, Mb catalase-like antioxidant activity remained unchanged under the same conditions. Notably, HOCl-modification of Mb decreased the rate of ferric-to-ferrous Mb reduction by a cytochrome b5 reductase system. Taken together, these data indicate oxidizing HOCl promotes Mb oxidation but not chlorination and that oxidized Mb shows altered Mb peroxidase-like activity and diminished rates of one-electron reduction by cytochrome b5 reductase, possibly affecting oxygen storage and transport however, Mb-catalase-like antioxidant activity remains unchanged.

The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 μM BAY11-7082 or vehicle (control) followed by SAA (10 μg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.

Determining the speciation of selenium in vivo is crucial to understanding the biological activity of this essential element, which is a popular dietary supplement due to its anti-cancer properties. Hyphenated techniques that combine separation and detection methods are traditionally and effectively used in selenium speciation analysis, but require extensive sample preparation that may affect speciation. Synchrotron-based X-ray absorption and fluorescence techniques offer an alternative approach to selenium speciation analysis that requires minimal sample preparation. We present a brief summary of some key HPLC-ICP-MS and ESI-MS/MS studies of the speciation of selenium in cells and rat tissues. We review the results of a top-down approach to selenium speciation in human lung cancer cells that aims to link the speciation and distribution of selenium to its biological activity using a combination of X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM). The results of this approach highlight the distinct fates of selenomethionine, methylselenocysteine and selenite in terms of their speciation and distribution within cells: organic selenium metabolites were widely distributed throughout the cells, whereas inorganic selenium metabolites were compartmentalized and associated with copper. New data from the XFM mapping of electrophoretically-separated cell lysates show the distribution of selenium in the proteins of selenomethionine-treated cells. Future applications of this top-down approach are discussed.

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.

Acute kidney injury causes significant morbidity and mortality in the community and clinic. Various pathologies, including renal and cardiovascular disease, traumatic injury/rhabdomyolysis, sepsis, and nephrotoxicity, that cause acute kidney injury (AKI), induce general or regional decreases in renal blood flow. The ensuing renal hypoxia and ischemia promotes the formation of reactive oxygen species (ROS) such as superoxide radical anions, peroxides, and hydroxyl radicals, that can oxidatively damage biomolecules and membranes, and affect organelle function and induce renal tubule cell injury, inflammation, and vascular dysfunction. Acute kidney injury is associated with increased oxidative damage, and various endogenous and synthetic antioxidants that mitigate source and derived oxidants are beneficial in cell-based and animal studies. However, the benefit of synthetic antioxidant supplementation in human acute kidney injury and renal disease remains to be realized. The endogenous low-molecular weight, non-proteinaceous antioxidant, ascorbate (vitamin C), is a promising therapeutic in human renal injury in critical illness and nephrotoxicity. Ascorbate may exert significant protection by reducing reactive oxygen species and renal oxidative damage via its antioxidant activity, and/or by its non-antioxidant functions in maintaining hydroxylase and monooxygenase enzymes, and endothelium and vascular function. Ascorbate supplementation may be particularly important in renal injury patients with low vitamin C status.

Oxidation of lipoproteins is thought to be an early event in atherogenesis. To evaluate whether aortic lipoprotein lipid (per)oxidation contributes to atherosclerosis, we investigated the time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E gene knockout (apoE–/–) mice receiving a high fat diet, and compared these changes with lesion development. Circulating buoyant lipoproteins and associated cholesterol (C), cholesteryl esters (CE), and α-tocopherol (α-TOH) increased within 1 month then remained largely constant up to 6 months. Coenzyme Q (CoQ) remained unchanged for the first 3 months and increased marginally after 6 months. With increasing duration of the diet, plasma lipids showed an increased propensity to undergo peroxyl radical-induced (per)oxidation. Absolute concentrations of aortic C, hydroperoxides and hydroxides of CE (CE-O(O)H) and α-TOH increased gradually while aortic CE increased more markedly with changes to cholesteryl linoleate being most pronounced. Aortic CoQ remained largely unchanged. Overall, the extent of aortic CE (per)oxidation remained low (≤1%) and the ratio of incremental changes of α-TOH to oxidizable lipid remained unchanged. Aortic biochemistry paralleled lesion formation, particularly that in the descending thoracic aorta. Together, our results show that progressing atherosclerosis in apoE–/– mice is associated with increased aortic lipid (per)oxidation as assessed by the concentrations of CE-O(O)H, measured directly by HPLC. This supports the oxidation theory. Measurement of aortic CE-O(O)H may be useful for mechanistic studies studying the relationship between inhibition of in vivo lipid (per)oxidation and atherosclerosis.—Letters, J. M., P. K. Witting, J. K. Christison, A. Weston Eriksson, K. Pettersson, and R. Stocker. Time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E knockout mice. J. Lipid Res. 1999. 40: 1104–1112.