A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli

This is the author accepted manuscript. The final version is available fromFerrata Storti Foundation via the DOI in this recordThe regulation of platelets by oxidants is critical for vascular health and may explain thrombotic complications in diseases such as diabetes and dementia, but remains poorly understood. Here, we describe a novel technique combining electron paramagnetic resonance spectroscopy and turbidimetry, which has been utilised to monitor simultaneously platelet activation and oxygen radical generation. This technique has been used to investigate the redox-dependence of human and mouse platelets. Using selective peptide inhibitors of NOXs on human platelets and genetically modified mouse platelets (NOX1-/- or NOX2-/-), we discovered that:1) intracellular but not extracellular superoxide anion generated by NADPH oxidases (NOXs) is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent activation; 3) NOX1 is the main source of oxygen radicals in response to collagen, while NOX2 is critical for activation by thrombin; 4) two platelet modulators, namely oxidised low density lipoproteins (oxLDL) and amyloid peptide β (Aβ), require activation of both NOX1 and NOX2 to pre-activate platelets. This study provides new insights on the redox dependence of platelet activation. It suggests the possibility of selectively inhibiting platelet agonists by targeting either NOX1 (for collagen) or NOX2 (for thrombin). Selective inhibition of either NOX1 or NOX2 impairs the potentiatory effect of tested platelet modulators (oxLDL and Aβ), but does not completely abolish platelet haemostatic function. This information offers new opportunities for the development of disease specific antiplatelet drugs with limited bleeding side effects by selectively targeting one NOX isoenzyme.British Heart Foundatio

NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation & vessel dysfunction and restore mouse hind-limb flow.

First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47phox, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BACKGROUND: Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH

Selective Light-Triggered Release of DNA from Gold Nanorods Switches Blood Clotting On and Off

Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.National Science Foundation (U.S.) (Grant DMR #0906838

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

Combined search for the quarks of a sequential fourth generation

Results are presented from a search for a fourth generation of quarks produced singly or in pairs in a data set corresponding to an integrated luminosity of 5 inverse femtobarns recorded by the CMS experiment at the LHC in 2011. A novel strategy has been developed for a combined search for quarks of the up and down type in decay channels with at least one isolated muon or electron. Limits on the mass of the fourth-generation quarks and the relevant Cabibbo-Kobayashi-Maskawa matrix elements are derived in the context of a simple extension of the standard model with a sequential fourth generation of fermions. The existence of mass-degenerate fourth-generation quarks with masses below 685 GeV is excluded at 95% confidence level for minimal off-diagonal mixing between the third- and the fourth-generation quarks. With a mass difference of 25 GeV between the quark masses, the obtained limit on the masses of the fourth-generation quarks shifts by about +/- 20 GeV. These results significantly reduce the allowed parameter space for a fourth generation of fermions.Comment: Replaced with published version. Added journal reference and DO

Search for New Physics with Jets and Missing Transverse Momentum in pp collisions at sqrt(s) = 7 TeV

A search for new physics is presented based on an event signature of at least three jets accompanied by large missing transverse momentum, using a data sample corresponding to an integrated luminosity of 36 inverse picobarns collected in proton--proton collisions at sqrt(s)=7 TeV with the CMS detector at the LHC. No excess of events is observed above the expected standard model backgrounds, which are all estimated from the data. Exclusion limits are presented for the constrained minimal supersymmetric extension of the standard model. Cross section limits are also presented using simplified models with new particles decaying to an undetected particle and one or two jets