Chronic Oxidative Stress Conditions Research Articles

Examining the Impact of Environmental and Genetic Factors that Influence Sarcospan Expression in the Heart

Objective: Susceptibility to cardiac injury can be hereditary and the most common causes include genetic variations in proteins regulating lipid metabolism, arterial hypertension, diabetes, body-mass index as well as environmental causes such as smoking. Sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has an important role in stabilizing the muscle membrane of striated muscle. Genetic variants in the SSPN gene have been linked with increased cardiovascular disease risk and studies are ongoing to identify underlying mechanisms. Our studies using perfused mouse hearts subjected to acute ischemia/reperfusion injury indicate that SSPN deletion increases infarct size and arrhythmia risk (Hwang et al, IJMS, 2023). In addition, adult SSPN−/− cardiomyocytes were found to have decreased antioxidative capacity and heightened susceptibility to oxidative stress. Therefore, we hypothesize that genetic factors that decrease cardiac SSPN expression will increase cardiac susceptibility of mice exposed to conditions of prolonged oxidative stress. In this study we examine the role of SSPN to heart injury caused by chronic oxidative stress mediated by cigarette smoke (CS). Methods: To assess the impact of chronic oxidative stress conditions on SSPN expression in the heart 2 month-old WT C57BL/6J mice were exposed to 4 months CS. We found that SSPN expression was significantly reduced the CS exposed hearts. Since CS increases oxidative damage and deteriorates antioxidant defenses of the heart and vascular system, we examined the combined impact of SSPN deficiency and chronic oxidative stress. To address this, 7-month-old WT and SSPN−/− male and female mice were exposed to CS for 4 months. The mice were assessed after CS for body composition (EchoMRI), glucose tolerance testing and echocardiography. Tissue histology, immunoblotting of oxidative and inflammatory stress markers, serum inflammatory markers were assessed. Lung fibroblasts were isolated from the smoked and non-smoked groups to evaluate inflammatory, fibrotic, and oxidative stress pathway activation to respective stimuli. Results: Preliminary findings indicate that after CS SSPN−/− hearts exhibit larger areas of myocardial damage than WT mice. Surprisingly, after CS the SSPN−/− mice showed better glucose tolerance than WT mice and no significant changes in body mass. Minor decrements were found in the cardiac function of the CSE mice. Exposure of lung fibroblasts obtained from non-smoked SSPN−/− mice to innate immune ligands revealed activation of specific pro-inflammatory pathways.Conclusions: SSPN-deficiency increases susceptibility to cardiac injury in mice subjected to CS exposure, likely through crosstalk mechanisms that exist between oxidative and inflammatory pathways that SSPN appears to influence. Funding Sources: Florida Department of Health, James and Esther King #21K12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial dysfunction as predictor of atherosclerosis during a translatitudinal sea voyage in the Arctic: prospective study

INTRODUCTION. Human labor activity in the Arctic takes place in severe climatic conditions associated with cooling, atmospheric pressure drops, high humidity, photoperiodicity, low oxygen content in the air. Endothelial dysfunction is an early pathophysiological sign and an independent predictor of poor prognosis in most cardiovascular diseases.
 OBJECTIVE.Тo analyze the dynamics of the level of metalloproteinase-9 and thrombin activity as predictors of the development of atherosclerosis in sailors during a translatitudinal sea voyage in the Arctic.
 MATERIALS AND METHODS. A prospective clinical and laboratory study of crew members was performed during the integrated marine research expedition “Transarctic-2019”. A clinical and laboratory study of the level of metalloprotease-9 (enzyme-linked immunosorbent assay), coagulation analysis of the parameters of the thrombin generation test, as well as ultrasonic dopplerography of the thickness of the intima-media complex were carried out. Statistical data processing was carried out using the methods of descriptive and analytical statistics in the StataCorp Stata 14.2 program. The study protocol was approved by the local ethical committee of the SSMU (protocol No. 03/5 dated May 27, 2015) and supported by the state task reg. No. NIOKTR 121030300111-7.
 RESULTS. A statistically significant increase in the level of MMP-9 in sailors after the expedition was noted, the relationship between the level of MMP-9, indicators of the thrombin generation test (lag time, min (p=0.0190), tPeak (p=0.0177), Peak (p= 0.0217) and KIM thickening.
 DISCUSSION. MMP-9 levels may be a predictor of early atherosclerosis and atherosclerotic plaque instability, as well as a risk factor for future adverse cardiovascular events. The relationship of MMP-9 with the kinetics of thrombin (one of the regulators of the antithrombotic state of the endothelium) during a translatitudinal voyage in sailors indicates the formation of readiness for prothrombotic events.
 CONCLUSION. Thus, in Arctic conditions, under conditions of chronic oxidative stress, which contribute to the progression of the development of endothelial dysfunction in the presence of external risk factors, the likelihood of progression of atherosclerotic vascular lesions increases, and, as a result, the development of vascular events.

Abstract 126: Defining how oxidative stress drives the evolution of aggressive breast cancers

Abstract Breast cancer is the most commonly diagnosed malignancy in women worldwide. It is a molecularly heterogeneous disease, which poses difficulties in the treatment of more advanced cancers. Despite this fact, the mechanisms contributing to the emergence of aggressive breast cancers remains poorly understood. We focus on reactive oxygen species (ROS), which are induced by a variety of stimuli in breast cancer cells. Moderately elevated ROS levels are tumor-promoting and facilitate the emergence of more aggressive tumours. Our laboratory has developed a unique model of HER2+ breast cancer that evolved to acquire more aggressive properties under conditions of chronic oxidative stress. We identified 20 transcription factors/co-regulators that were specifically overexpressed in these aggressive breast cancers, compared to their parental counterparts. We hypothesize that these transcription factors are central to the ability of breast tumors to adapt to chronic oxidative stress and acquire more aggressive properties. To test this, I have performed an in vivo functional shRNA screen to identify those transcriptional regulators that drive an aggressive phenotype. This will be followed by functional validation studies to interrogate whether and how these unique transcription factors overexpressed in aggressive breast cancers impact tumour growth, metastasis, and drug resistance. Mechanistically, we will explore whether and how these transcriptional responses mediate adaptive responses to tumor microenvironmental stressors, including altered metabolism, hypoxia, and tumor immune responses. This research will broaden our understanding of how breast cancers adapt to oxidative stress responses and represents a necessary first step in the development of novel therapeutics against these invulnerable malignancies. Citation Format: Caitlynn N. Mirabelli, Rachel La Selva, John Heath, Steven Hébert, Jutta Steinberger, Jialin Jiang, Claudia Kleinman, Sidong Huang, Josie Ursini-Siegel. Defining how oxidative stress drives the evolution of aggressive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 126.

Potential Effects of Resveratrol on Obesity Markers of Iraqi Women

Obesity is defined as a condition of chronic oxidative stress and chronic systemic inflammation, and it is associated with many health problems. Resveratrol is a polyphenolic compound noted to exert beneficial effects on various diseases such as cardiovascular diseases, atherosclerosis, diabetes mellitus, and nephropathy. The healthful effects of resveratrol are thought to be attributed to its antioxidant properties. In this interventional prospective randomized controlled trial, female patients diagnosed with obesity according to the WHO criteria and were randomly allocated to either control or resveratrol group. Resveratrol supplement showed a significant reduction in weight, body mass index (BMI) and waist circumference (WC). From the above results, one can conclude that oral supplementation of resveratrol can improve Anthropometric parameters.

Molecular Mechanisms of ER Stress and UPR in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease involving aggregation of misfolded proteins inside the neuron causing prolonged cellular stress. The neuropathological hallmarks of AD include the formation of senile plaques and neurofibrillary tangles in specific brain regions that lead to synaptic loss and neuronal death. The exact mechanism of neuron dysfunction in AD remains obscure. In recent years, endoplasmic reticulum (ER) dysfunction has been implicated in neuronal degeneration seen in AD. Apart from AD, many other diseases also involve misfolded proteins aggregations in the ER, a condition referred to as ER stress. The response of the cell to ER stress is to activate a group of signaling pathways called unfolded protein response (UPR) that stimulates a particular transcriptional program to restore ER function and ensure cell survival. ER stress also involves the generation of reactive oxygen species (ROS) that, together with mitochondrial ROS and decreased effectiveness of antioxidant mechanisms, producing a condition of chronic oxidative stress. The unfolded proteins may not always produce a response that leads to the restoration of cellular functions, but they may also lead to inflammation by a set of different pathways with deleterious consequences. In this review, we extensively discuss the role of ER stress and how to target it using different pharmacological approaches in AD development and onset.

MG53 Interacts with Cardiolipin to Protect Mitochondria from Ischemia-Reperfusion Induced Oxidative Stress

MG53 is a tripartite motif protein that is essential to plasma membrane regeneration. It binds to phosphatidylserine and facilitates the formation of repair patches at sites of plasma membrane disruption. Our previous studies have reported MG53's efficacy in ameliorating ischemia-reperfusion (I/R) injury to multiple organs including the heart, lungs, kidney, and brain. In I/R injury, mitochondria, however, are the primary victim of damage and the exposure of mitochondrial cardiolipin (CL) in response to I/R-induced oxidative stress procures cell death. Thus far, the role of MG53 in the preservation of mitochondrial function has not been studied. Here, we test the hypothesis that MG53 interacts with CL to protect mitochondria from oxidative stress. We find that mitochondria in the heart of MG53 knockout mouse are more susceptible to I/R injury than those of wild type mice. We also observe that MG53 targets mitochondria under chronic oxidative stress conditions, such as high-fat diet induced metabolic syndrome and amyotrophic lateral sclerosis. We demonstrate that MG53 binds to CL through a lipid dot-blot and a quantitative ELISA lipid-protein binding assay. Additionally, we show that exogenous recombinant human MG53 (rhMG53) protein can be up-taken by a variety of cells, including mesenchymal stem cells, human corneal epithelial cells, and valvular interstitial cells. Once internalized, rhMG53 translocates to mitochondria in response to oxidative stress induced by anoxia-reoxygenation treatment as revealed by live-cell imaging and 3D reconstruction of confocal microscopy. These data suggest that MG53 can target damaged mitochondria through a potential CL signaling mechanism and may implicate MG53 in mitochondrial membrane protection. To our knowledge, this study is the first to explore the interaction of MG53 with subcellular structures and provides a novel mechanism for MG53-mediated amelioration of I/R-induced tissue injury.

PP69 - High-glucose-induced impairment of the proteasomal system in pancreatic Min6 cells

Protein degradation by the 26S proteasome is a fundamental process involved in a broad range of cellular activities, yet how proteasome activity is regulated remains poorly understood. In particular, the mechanisms of redox-dependent regulation of the ubiquitin-proteasomal system (UPS) are not clear. High glucose has been proposed to promote oxidative stress in different cellular models. Min6 b-cells grown under high glucose conditions showed a decreased 20S (chemotrypsin) activity compared to control cells, while 20S core subunits were increased as determined by western blot. Peroxiredoxin 5 levels were increased under these conditions, suggesting a response to chronic oxidative stress conditions. On the other hand, short-term exposure of the cells to high glucose concentrations caused a decrease in 26S activity and an increase in 20S activity. Similar results were observed when cells were incubated with the mitochondrial uncoupler antimycin A or hydrogen peroxide. Glucose-induced increase of 20S activity was time-dependent and this effect was abolished when the 20S activity was measured in the presence of the thiol reducing agent dithiotreitol. Dimerization of mitochondrial and cytosolic peroxiredoxins evidenced an increase in the steady-state concentration of peroxides under these conditions. Moreover, non-denaturing PAGE followed by in-gel proteasome activity evidenced the formation of aggregates and changes in proteasome structure which were partially reversed by thiol reduction. Our data suggest that high glucose promotes oxidative stress in Min6 b-cells with a concomitant UPS impairment. Further studies will focus on the thiol modifications of the UPS induced under this conditions and its reversibility.

Accelerated Aging during Chronic Oxidative Stress: A Role for PARP-1

Oxidative stress plays a major role in the pathophysiology of chronic inflammatory disease and it has also been linked to accelerated telomere shortening. Telomeres are specialized structures at the ends of linear chromosomes that protect these ends from degradation and fusion. Telomeres shorten with each cell division eventually leading to cellular senescence. Research has shown that poly(ADP-ribose) polymerase-1 (PARP-1) and subtelomeric methylation play a role in telomere stability. We hypothesized that PARP-1 plays a role in accelerated aging in chronic inflammatory diseases due to its role as coactivator of NF-κb and AP-1. Therefore we evaluated the effect of chronic PARP-1 inhibition (by fisetin and minocycline) in human fibroblasts (HF) cultured under normal conditions and under conditions of chronic oxidative stress, induced by tert-butyl hydroperoxide (t-BHP). Results showed that PARP-1 inhibition under normal culturing conditions accelerated the rate of telomere shortening. However, under conditions of chronic oxidative stress, PARP-1 inhibition did not show accelerated telomere shortening. We also observed a strong correlation between telomere length and subtelomeric methylation status of HF cells. We conclude that chronic PARP-1 inhibition appears to be beneficial in conditions of chronic oxidative stress but may be detrimental under relatively normal conditions.

Abnormal isoaspartyl residues in erythrocyte membranes from psoriatic patients

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function and can trigger autoimmune responses. To minimize the damage, IsoAsp can be "repaired" by a specific L-isoaspartate-(D-aspartate)-protein-O-methyltransferase. The condition of chronic oxidative stress reported in psoriatic patients, and the potential etiological role of unknown self-antigens, prompted us to investigate Asn deamidation in psoriatic tissues. Erythrocytes (RBC) were selected as the model system since, lacking protein synthesis apparatus, they are unable to replace damaged proteins. Blood samples were obtained from 36 patients and 34 controls. L-isoAsp content was highly increased in RBC membrane proteins from psoriatic patients. Deamidated species included ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3. A functional analysis demonstrated that this result was unrelated to a reduced efficiency of the S-adenosylmethionine-dependent repair system suggesting an increased protein instability at Asn sites, responsible for IsoAsp accumulation in psoriatic patients.

Accelerated aging during chronic oxidative stress: a role for PARP‐1?

Oxidative stress plays a major role in the pathophysiology of chronic inflammatory diseases, but it has also been linked to accelerated telomere shortening. Telomeres are specialized structures at the ends of linear chromosomes that protect these ends from degradation and fusion. Loss of telomeric DNA will result in telomere shortening and eventually cellular senescence. Research has shown that poly(ADP‐ribose)‐polymerase‐1 (PARP‐1) plays a role in telomere length regulation. We hypothesized that PARP‐1 plays a role in accelerated aging in chronic inflammatory diseases. In cultured human fibroblasts, effects of mild and strong inhibition of PARP‐1 under conditions of chronic oxidative stress, induced by tert‐butyl hydroperoxide (t‐BHP), were investigated. Fisetin (Fis), a flavonoid, was used as a mild inhibitor and Minocycline (Mino), an antibiotic, as a strong inhibitor of PARP‐1. Results indicated that t‐BHP and Mino alone induced a delay in cell growth and accelerated telomere shortening. However, when Mino was given in combination with t‐BHP, cells did not show a decrease in telomere length. Additionally, both Fis and Mino lowered the transcription of inflammation‐related genes in cells treated with t‐BHP. We conclude that PARP‐1 inhibition appears to be beneficial in conditions of chronic oxidative stress, but may be detrimental under relatively normal conditions.

Oxidative stress and endosome recycling are complementary mechanisms reorganizing the T-cell receptor signaling complex in SLE

Oxidative stress and endosome recycling are complementary mechanisms reorganizing the T-cell receptor signaling complex in SLE

Genistein up-regulates the iron efflux system in glial cells

Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological disorders. The soybean isoflavone genistein possesses antioxidant properties and selective estrogen-like activities. Here, a possible role of genistein in modulation of iron transport was explored in glial cells. Genistein significantly increased iron export through estrogen receptor-β-dependent p38 MAPK activation. Evidence is presented that this effect is associated to a p38 MAPK-triggered up-regulation of the iron export system made by ceruloplasmin and ferroportin-1, a pathway requiring activation of the transcription factor C/EBP.

Possible benefits of micronutrient supplementation in the treatment and management of HIV infection and AIDS

Recently, several reports have indicated that individuals living with HIV/AIDS undergo a condition of chronic oxidative stress with a resultant decline in nutritional antioxidants and other micronutrients. It has also been reported that these micronutrient deficiencies interfere with immune functions, weaken epithelial integrity, contribute to oxidative stress and enhance HIV disease progression. Reports from observational studies have led to an increasing interest in the possible benefits of micronutrient supplementation as a cost-effective strategy for improving oxidative and nutritional status. Micronutrient supplementation may also assist in the possible prevention of vertical transmission of HIV from mother to child especially in low-income countries where antiretroviral therapy and prophylactic drugs are not readily available. However, there are conflicting reports from other observational studies as to the usefulness or benefits of micronutrient supplementation in the treatment and management of HIV/AIDS. In this review we examine possible benefits of micronutrient supplementation in the treatment and management of HIV infection and AIDS. Key words: Vitamins, antioxidants, micronutrients, supplementation, HIV infection, AIDS.

Plant adaptogens increase lifespan and stress resistance in C. elegans

Extracts of plant adaptogens such as Eleutherococcus senticosus (or Acanthopanax senticosus) and Rhodiola rosea can increase stress resistance in several model systems. We now show that both extracts also increase the mean lifespan of the nematode C. elegans in a dose-dependent way. In at least four independent experiments, 250 microg/ml Eleutherococcus (SHE-3) and 10-25 microg/ml Rhodiola (SHR-5) significantly increased life span between 10 and 20% (P < 0.001), increased the maximum lifespan with 2-3 days and postponed the moment when the first individuals in a population die, suggesting a modulation of the ageing process. With higher concentrations, less effect was observed, whereas at the highest concentrations tested (2500 microg/ml Eleutherococcus and 250 microg/ml Rhodiola) a lifespan shortening effect was observed of 15-25% (P < 0.001). Both adaptogen extracts were also able to increase stress resistance in C. elegans: against a relatively short heat shock (35 degrees C during 3 h) as well as chronic heat treatment at 26 degrees C. An increase against chronic oxidative stress conditions was observed in mev-1 mutants, and during exposure of the wild type nematode to paraquat (10 mM) or UV stress, be it less efficiently. Concerning the mode of action: both adaptogens induce translocation of the DAF-16 transcription factor from the cytoplasm into the nucleus, suggesting a reprogramming of transcriptional activities favoring the synthesis of proteins involved in stress resistance (such as the chaperone HSP-16) and longevity. Based on these observations, it is suggested that adaptogens are experienced as mild stressors at the lifespan-enhancing concentrations and thereby induce increased stress resistance and a longer lifespan.

Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress

Gaucher disease (GD) is a lysosomal storage disorder, due to glucosylceramide (GlcCer) accumulation in several body tissues, which causes cellular failure by yet unidentified mechanisms. Several evidence indicates that GD pathogenesis is associated to an impairment in intracellular redox state. In fibroblast primary cultures, reactive oxygen species (ROS) levels and protein carbonyl content resulted significantly increased in GD patients compared to healthy donors, suggesting that GD cells, facing a condition of chronic oxidative stress, have evolved an adaptive response to survive. The ROS rise is probably due to NAD(P)H oxidase activity, being inhibited by the treatment with diphenylene iodonium chloride. Interestingly, GD cells are more sensitive to H(2)O(2) induced cell death, suggesting a dysregulation in the adaptive response to oxidative stress in which APE1/Ref-1 plays a central role. We found that the cytoplasmic amounts of APE1/Ref-1 protein were significantly higher in GD fibroblasts with respect to controls, and that GD cells failed to upregulate its expression upon H(2)O(2) treatment. Both ROS and APE1/Ref-1 increases are due to GlcCer accumulation, being prevented by treatment of GD fibroblasts with Cerezyme and induced in healthy fibroblasts treated with conduritol-beta-epoxide. These data, suggesting that GD cells display an impairment in the cellular redox state and in the adaptive cellular response to oxidative stress, may open new perspectives in the comprehension of GD pathogenesis.

Up‐regulation of P‐glycoprotein expression by glutathione depletion‐induced oxidative stress in rat brain microvessel endothelial cells

Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier (BBB) are subjected to chronic oxidative stress. In this study, we investigated the effect of such stress, produced with the GSH synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO), on expression of P-glycoprotein (Pgp) in primary cultured rat brain microvessel endothelial cells that comprise the blood-brain barrier (BBB). Application of BSO to cell monolayers at concentrations up to 800 microm caused increases in expression of Pgp. Concentrations >or= 400 microm BSO decreased cell viability. Application of 200 microm BSO caused a significant increase in Pgp function activity, as assessed by rhodamine 123 (Rh123) accumulation experiments. At this concentration, BSO produced time-dependent decreases in levels of intracellular GSH and increases in levels of intracellular reactive oxygen species (iROS). The increases were also observed within 48 h following BSO treatment in mdr1a and mdr1b mRNA. Exposure of cells to BSO for 24 h produced maximal effects in the accumulation of iROS, and in expression and function of Pgp. The ROS scavenger N-acetylcysteine prevented ROS generation and attenuated the changes of both expression and activity of Pgp induced by BSO. Therefore, the transport of Pgp substrates may be affected by changing Pgp expression under conditions of chronic oxidative stress induced by GSH depletion.

Ataxia Telangiectasia Mutated (ATM) and ATM and Rad3-related Protein Exhibit Selective Target Specificities in Response to Different Forms of DNA Damage

The ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3-related) protein kinases exert cell cycle delay, in part, by phosphorylating Checkpoint kinase (Chk) 1, Chk2, and p53. It is well established that ATR is activated following UV light-induced DNA damage such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones, whereas ATM is activated in response to double strand DNA breaks. Here we clarify the activation of these kinases in cells exposed to IR, UV, and hyperoxia, a condition of chronic oxidative stress resulting in clastogenic DNA damage. Phosphorylation on Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following oxidative damage by IR involved both ATM and ATR. In response to ultraviolet radiation-induced stalled replication forks, phosphorylation on Chk1 and p53 required ATR, whereas Chk2 required ATM. Cells exposed to hyperoxia exhibited growth delay in G1, S, and G2 that was disrupted by wortmannin. Consistent with ATM or ATR activation, hyperoxia induced wortmannin-sensitive phosphorylation of Chk1, Chk2, and p53. By using ATM- and ATR-defective cells, phosphorylation on Chk1, Chk2, and p53 was found to be ATM-dependent, whereas ATR also contributed to Chk1 phosphorylation. These data reveal activated ATM and ATR exhibit selective substrate specificity in response to different genotoxic agents.

Cigarette smoke triggers macrophage adhesion and activation: role of lipid peroxidation products and scavenger receptor.

Pulmonary emphysema in chronic obstructive pulmonary disease (COPD) is characterized by the destruction of the alveolar walls leading to permanent enlargement of distal respiratory air spaces. A major causal factor is cigarette smoking, which produces conditions of chronic oxidative stress within the lungs. At a cellular level, increased macrophage accumulation and retention within the alveolar interstitial spaces is pivotal to the development of emphysema. To date it has been unclear as to the underlying mechanisms relating chronic oxidative stress to macrophage accumulation and retention. Our study was initiated to ascertain the role of modification of extracellular matrix proteins with cigarette smoke and products of lipid peroxidation on macrophage adhesion and activation. Increased numbers of macrophages were seen adhering to cigarette smoke-modified collagen IV as compared to unmodified collagen, where little or no adherent macrophages were observed. Similar observations were made when collagen was modified with either acrolein or 4-hydroxy-2-nonenal. Adhesion could be blocked with either fucoidan or a monoclonal antibody against the Type A macrophage scavenger receptor. Also, modified collagen triggered both oxidative burst and MCP-1 release in macrophages. These results, therefore, highlight a potential mechanism by which oxidative stress through the production of reactive carbonyls promotes macrophage accumulation, retention, and activation, independently of other proinflammatory stimuli. The implications of this for the development of emphysema in COPD are discussed.

The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite.

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.

Oxidized LAT and Hyporesponsiveness

Correct localization to lipid rafts is essential for LAT (linker for activation of T cells)-mediated signaling through the T cell antigen receptor (TCR). Under conditions of chronic oxidative stress, T cells are hyporesponsive to stimuli. For example, in rheumatoid arthritis (RA), T cell insensitivity is thought to contribute to the disorder. Grinhuis et al . found that LAT localization in T cells is perturbed during chronic oxidative stress and results in decreased signaling outputs. LAT isolated from T cells treated with an inhibitor of glutathione (GSH) synthesis exhibited faster migration than LAT from untreated T cells under nonreducing conditions in gel electrophoresis. This suggested that LAT might form intramolecular disulfide bonds under conditions of chronic oxidative stress. Mutational analysis of Cys residues on LAT revealed that C117S or C26/29S mutants were partially insensitive to chronic oxidative stress (presumably because they could not become oxidized and form disulfide bridges), as shown by their continued presence in lipid rafts and by the ability of cells expressing these mutants to stimulate gene expression from interleukin-2 promoter-driven reporter genes. Oxidative stress also greatly reduced the TCR- and LAT-dependent activation of the transcription factors AP-1, NFAT, and NF-κB. However, expression of oxidation-insensitive LAT mutants in T cells partially reversed this inhibition. The authors suggest that the transmembrane helix of LAT might be sufficiently disrupted by chronic oxidative conditions such that LAT cannot remain localized to the lipid rafts. Other data suggested that disulfide bonds might form between Cys117 and probably Cys29. The data suggest that T cell hyporesponsiveness in chronic oxidative conditions results, in part, from improper localization of wild-type LAT, and thus ineffective signaling. For an excellent review on the function of the LAT protein, see the review by Wange . S. I. Gringhuis, E. A. M. Papendrecht-van der Voort, A. Leow, E. W. N. Levarht, F. C. Breedveld, C. L. Verweij, Effect of redox balance alterations on cellular localization of LAT and downstream T-cell receptor signaling pathways. Mol. Cell. Biol. 22 , 400-411 (2002). [Abstract] [Full Text] R. L. Wange, LAT, the linker for activation of T cells: A bridge between T cell-specific and general signaling pathways. Science's STKE (2000), http://www.stke.org/cgi/content/full/OC_sigtrans;2000/63/re1. [Abstract] [Full Text]