Oxidative Stress Is Associated with an Increased Antioxidant Defense in Elderly Subjects: A Multilevel Approach

Background

Studies of associations between plasma GSH-Px activity and cardiovascular risk factors have been done in humans, and contradictory results have been reported. The aim of our study was to assess the association between the scavenger antioxidant enzyme glutathione peroxidase (GSH-Px) activity in plasma and the presence of novel and classical cardiovascular risk factors in elderly patients.

Methods

We performed a cross-sectional study with baseline data from a subsample of the PREDIMED (PREvención con DIeta MEDiterránea) study in Spain. Participants were 1,060 asymptomatic subjects at high risk for cardiovascular disease (CVD), aged 55 to 80, selected from 8 primary health care centers (PHCCs). We assessed classical CVD risk factors, plasma oxidized low-density lipoproteins (ox-LDL), and glutathione peroxidase (GSH-Px) using multilevel statistical procedures.

Results

Mean GSH-Px value was 612 U/L (SE: 12 U/L), with variation between PHCCs ranging from 549 to 674 U/L (Variance = 1013.5; P<0.001). Between-participants variability within a PHCC accounted for 89% of the total variation. Both glucose and oxidized LDL were positively associated with GSH-Px activity after adjustment for possible confounder variables (P = 0.03 and P = 0.01, respectively).

Conclusion

In a population at high cardiovascular risk, a positive linear association was observed between plasma GSH-Px activity and both glucose and ox-LDL levels. The high GSH-Px activity observed when an oxidative stress situation occurred, such as hyperglycemia and lipid oxidative damage, could be interpreted as a healthy defensive response against oxidative injury in our cardiovascular risk population.     

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.     

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.     

Impact of Virgin Olive Oil and Phenol-Enriched Virgin Olive Oils on the HDL Proteome in Hypercholesterolemic Subjects: A Double Blind,Randomized, Controlled,Cross-Over Clinical Trial (VOHF Study)

The effects of olive oil phenolic compounds (PCs) on HDL proteome, with respect to new aspects of cardioprotective properties, are still unknown. The aim of this study was to assess the impact on the HDL protein cargo of the intake of virgin olive oil (VOO) and two functional VOOs, enriched with their own PCs (FVOO) or complemented with thyme PCs (FVOOT), in hypercholesterolemic subjects. Eligible volunteers were recruited from the IMIM-Hospital del Mar Medical Research Institute (Spain) from April 2012 to September 2012. Thirty-three hypercholesterolemic participants (total cholesterol >200mg/dL; 19 men and 14 women; aged 35 to 80 years) were randomized in the double-blind, controlled, cross-over VOHF clinical trial. The subjects received for 3 weeks 25 mL/day of: VOO, FVOO, or FVOOT. Using a quantitative proteomics approach, 127 HDL-associated proteins were identified. Among these, 15 were commonly differently expressed after the three VOO interventions compared to baseline, with specific changes observed for each intervention. The 15 common proteins were mainly involved in the following pathways: LXR/RXR activation, acute phase response, and atherosclerosis. The three VOOs were well tolerated by all participants. Consumption of VOO, or phenol-enriched VOOs, has an impact on the HDL proteome in a cardioprotective mode by up-regulating proteins related to cholesterol homeostasis, protection against oxidation and blood coagulation while down-regulating proteins implicated in acute-phase response, lipid transport, and immune response. The common observed protein expression modifications after the three VOOs indicate a major matrix effect.

Trial Registration

International Standard Randomized Controlled Trials ISRCTN77500181.     

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys¹³³ and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor α production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation.TLR4 is a receptor for lipopolysaccharide (LPS)⁴ a major constituent of outer membrane of Gram-negative bacteria. MD-2 is the final LPS-binding protein in the recognition cascade before TLR4 transmits the signal across the cell membrane to activate the inflammatory response. MD-2 binds to the ectodomain of TLR4 and binds LPS either alone or in complex with TLR4 (1, 2). Mice deficient in MD-2 survive endotoxic shock (3). MD-2 has been indispensable in almost all investigated conditions of TLR4-triggered inflammation; therefore, it could represent the “Achilles'' heel” of the inflammatory response to LPS and a target for a pharmacological intervention in endotoxemia as well as other conditions involving cell activation mediated by TLR4 (4, 5). The existence of a single free cysteine residue among the seven cysteine residues has been predicted from MD-2 mutagenesis (6, 7) and molecular modeling proposed that Cys¹³³ lies in the hydrophobic pocket (8, 9). The hydrophobic binding site of MD-2 was also mapped by an apolar probe, bis-ANS, which does not contain acyl chains as most LPS antagonists yet preserves the characteristic structural motif of lipid A, consisting of a hydrophobic region and a pair of separated negatively charged groups (10). Crystal structures of MD-2 with bound eritoran or lipid IVa confirmed the location of Cys¹³³ in the hydrophobic pocket in close vicinity of bound lipid A derivatives (11, 12). The free cysteine residue inside the binding pocket can thus be a target for irreversible inhibition of MD-2 activity. An inhibitory mechanism based on a covalent modification of a free cysteine residue in the active or binding site of a protein has been demonstrated for other proteins, such as in cysteine proteases, where the cysteine residue participates in the catalytic triad (13), in cholesteryl ester transfer protein (CETP) (14), IκB kinase (15), thioredoxin reductase (16), and sortase (17).In our study, we investigated the possibility of targeting free cysteine residue of MD-2 for the inhibition of LPS signaling. We determined covalent binding into the hydrophobic pocket of MD-2 for fluorescent compounds 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and N-pyrene maleimide. Drugs JTT-705 and auranofin, already in use for alternative indications, were also shown to bind to MD-2 and decrease LPS signaling. The identity of Cys¹³³ as the residue responsible for this interaction was demonstrated by point mutagenesis. Our results confirm that the proposed mechanism of inhibition of MD-2 can have potential therapeutic value but may also have a physiological role.     

Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.     

Middle Eocene rodents from Peruvian Amazonia reveal the pattern and timing of caviomorph origins and biogeography

The long-term isolation of South America during most of the Cenozoic produced a highly peculiar terrestrial vertebrate biota, with a wide array of mammal groups, among which caviomorph rodents and platyrrhine primates are Mid-Cenozoic immigrants. In the absence of indisputable pre-Oligocene South American rodents or primates, the mode, timing and biogeography of these extraordinary dispersals remained debated. Here, we describe South America's oldest known rodents, based on a new diverse caviomorph assemblage from the late Middle Eocene (approx. 41 Ma) of Peru, including five small rodents with three stem caviomorphs. Instead of being tied to the Eocene/Oligocene global cooling and drying episode (approx. 34 Ma), as previously considered, the arrival of caviomorphs and their initial radiation in South America probably occurred under much warmer and wetter conditions, around the Mid-Eocene Climatic Optimum. Our phylogenetic results reaffirm the African origin of South American rodents and support a trans-Atlantic dispersal of these mammals during Middle Eocene times. This discovery further extends the gap (approx. 15 Myr) between first appearances of rodents and primates in South America.     

Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF

Standard treatments for erectile dysfunction (ED) (i.e., PDE5 inhibitors) are less effective in diabetic patients for unknown reasons. Endothelium-dependent relaxation (EDR) of human corpus cavernosum (HCC) depends on nitric oxide (NO), while in human penile resistance arteries (HPRA) endothelium-derived hyperpolarizing factor (EDHF) and NO participate. Here we show that diabetes significantly reduced EDR induced by acetylcholine (ACh) in HCC and HPRA. Relaxation attributed to EDHF was also impaired in HPRA from diabetic patients. The PDE5 inhibitor, sildenafil (10nM), reversed diabetes-induced endothelial dysfunction in HCC, but not in HPRA. Calcium dobesilate (DOBE; 10 microM) fully reversed diabetes-induced endothelial dysfunction in HPRA by specifically potentiating the EDHF-mediated component of EDR. Impairment by diabetes of NO and EDHF-dependent responses precluded the complete recovery of endothelial function in HPRA by sildenafil. This could explain the poor clinical response to PDE5 inhibitors of diabetic men with ED and suggests that a pharmacological approach that combines enhancement of NO/cGMP and EDHF pathways could be necessary to treat ED in many diabetic men.     

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.