AB5830 Sigma-AldrichAnti-8-Hydroxydeoxyguanosine Antibody

Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2-26 (Ac2-26). Collagen IV (Col IV)-targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr(-/-) mice. When administered to mice with preexisting lesions, Col IV-Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.

Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell type-specific causation studies in vivo are lacking, and the molecular mechanisms of potential proatherogenic effects remain to be determined.Our aims were to assess the importance of macrophage mitoOS in atherogenesis and to explore the underlying molecular mechanisms.We first validated Western diet-fed Ldlr(-/-) mice as a model of human mitoOS-atherosclerosis association by showing that non-nuclear oxidative DNA damage, a marker of mitoOS in lesional macrophages, correlates with aortic root lesion development. To investigate the importance of macrophage mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6c(hi) monocyte infiltration into lesions, and lower levels of monocyte chemotactic protein-1. The decrease in lesional monocyte chemotactic protein-1 was associated with the suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the proinflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed monocyte chemotactic protein-1 expression by decreasing the activation of the IκB-kinase β-RelA NF-κB pathway.MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.

Selenium (Se) is a crucial element exerting antioxidant and neuroprotective effects in different toxic models. It has been suggested that Se acts through selenoproteins, of which thioredoxin reductase (TrxR) is relevant for reduction of harmful hydroperoxides and maintenance of thioredoxin (Trx) redox activity. Of note, the Trx/TrxR system remains poorly studied in toxic models of degenerative disorders. Despite previous reports of our group have demonstrated a protective role of Se in the excitotoxic/pro-oxidant model induced by quinolinic acid (QUIN) in the rat striatum (Santamaría et al., 2003, 2005), the precise mechanism(s) by which Se is inducing protection remains unclear. In this work, we characterized the time course of protective events elicited by Se as pretreatment (Na(2)SO(3), 0.625mg/kg/day, i.p., administered for 5 consecutive days) in the toxic pattern produced by a single infusion of QUIN (240nmol/μl) in the rat striatum, to further explore whether TrxR is involved in the Se-induced protection and how is regulated. Se attenuated the QUIN-induced early reactive oxygen species formation, lipid peroxidation, oxidative damage to DNA, loss of mitochondrial reductive capacity and morphological alterations in the striatum. Our results also revealed a novel pattern in which QUIN transiently stimulated an early TrxR cellular localization/distribution (at 30min and 2h post-lesion, evidenced by immunohistochemistry), to further stimulate a delayed protein activation (at 24h) in a manner likely representing a compensatory response to the oxidative damage in course. In turn, Se induced an early stimulation of TrxR activity and expression in a time course that matches with the reduction of the QUIN-induced oxidative damage, suggesting that the Trx/TrxR system contributes to the resistance of nerve tissue to QUIN toxicity.

It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

Sestrin-2 is involved in p53-dependent antioxidant defenses and in the maintenance of metabolic homeostasis. We hypothesize that sestrin-2 expression is altered in the brains of subjects diagnosed with human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) due to neuronal oxidative stress. We studied sestrin-2 immunoreactivity in 42 isocortex sections from HIV-1-infected subjects compared to 18 age-matched non-HIV controls and 19 advanced Alzheimer's disease (AD) cases. With HIV infection, the sestrin-2 immunoreactivity pattern shifted from neuropil predominance (N) to neuropil and neuronal-soma co-dominance (NS) and neuronal-soma predominance (S; P less than  0.0001, Chi-square test for linear trend). Among HIV cases showing the NS or S pattern, HAND cases were preferentially associated with the S pattern (n = 10 of 20) compared to cognitively intact cases (n = 1 of 11; P = 0.047, Fisher's exact test). In AD brains, sestrin-2 immunoreactivity was mostly intense in the neuropil and co-localized with phospho-Tau immunoreactivity in a subset of neurofibrillary lesions. Phospho-Tau-immunoreactive neurofibrillary lesions were rare in HIV cases and their occurrence was not associated with HAND. Levels of isocortical 8-hydroxy-deoxyguanosine (marker of nucleic acid oxidation) immunoreactivity were not significantly altered in HAND cases compared to cognitively intact HIV cases. In conclusion, the sestrin-2 immunoreactivity redistribution to neuronal soma in HAND suggests unique involvement of sestrin-2 in the pathophysiology of HAND, which is different from the role of sestrin-2 in AD pathogenesis. Alternatively, the difference in sestrin-2 immunoreactivity distribution between HAND and AD may be related to different degrees of severity or stages of oxidative stress.

Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal.

Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5(-/-)) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5(-/-) animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on beta1 integrins. These results were validated in Fbln5(RGE/RGE) mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5(RGE/RGE) mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

Tandem repeat expansion is responsible for the Repeat Expansion Diseases, a group of human genetic disorders that includes Fragile X syndrome (FXS). FXS results from expansion of a premutation (PM) allele having 55-200 CGG.CCG-repeats in the 5' UTR of the FMR1 gene. The mechanism of expansion is unknown. We have treated FX PM mice with potassium bromate (KBrO(3)), a potent DNA oxidizing agent. We then monitored the germline and somatic expansion frequency in the progeny of these animals. We show here that KBrO(3) increased both the level of 8-oxoG in the oocytes of treated animals and the germline expansion frequency. Our data thus suggest that oxidative damage may be a factor that could affect expansion risk in humans.

This study aims to demonstrate the role of stress-induced senescence in aged-related neointimal formation. We demonstrated that aging increases senescence-associated beta-galactosidase activity (SA-beta-Gal) after vascular injury and the subsequent neointimal formation (neointima-to-media ratio: 0.8 +/- 0.2 vs. 0.54 +/- 0.15) in rats. We found that senescent cells (SA-beta-Gal(+) p21(+)) were scattered throughout the media and adventitia of the vascular wall at day 7 after injury and reached their maximum number at day 14. However, senescent cells only persisted in the injured arteries of aged animals until day 30. No senescent cells were observed in the noninjured, contralateral artery. Interestingly, vascular senescent cells accumulated genomic 8-oxo-7,8-dihydrodeoxyguanine, indicating that these cells were under intense oxidative stress. To demonstrate whether senescence worsens intimal hyperplasia after injury, we seeded matrigel-embedded senescent and nonsenescent vascular smooth muscle cells around injured vessels. The neointima was thicker in arteries treated with senescent cells with respect to those that received normal cells (neointima-to-media ratio: 0.41 +/- 0.105 vs. 0.26 +/- 0.04). In conclusion, these results demonstrate that vascular senescence is not only a consequence of postinjury oxidative stress but is also a worsening factor for neointimal development in the aging vasculature.

Suppression of the renin-angiotensin system has proven efficacy for mitigation and treatment of radiation nephropathy, and it has been hypothesized that this efficacy is due to suppression of radiation-induced chronic oxidative stress. It is known that radiation exposure leads to acute oxidative stress, but direct evidence for radiation-induced chronic renal oxidative stress is sparse. We looked for evidence of oxidative stress after total-body irradiation in a rat model, focusing on the period before there is physiologically significant renal damage. No statistically significant increase in urinary 8-isoprostane (a marker of lipid peroxidation) or carbonylated proteins (a marker of protein oxidation) was found over the first 42 days after irradiation, while a small but statistically significant increase in urinary 8-hydroxydeoxy-guanosine (a marker of DNA oxidation) was detected at 35-55 days. When we examined renal tissue from these animals, we found no significant increase in either DNA or protein oxidation products over the first 89 days after irradiation. Using five different standard methods for detecting oxidative stress in vivo, we found no definitive evidence for radiation-induced renal chronic oxidative stress. If chronic oxidative stress is part of the pathogenesis of radiation nephropathy, it does not leave widespread or easily detectable evidence behind.