1 Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET-1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24 h with ET-1 (10 pM-10 nM) in the absence or presence of the ET B receptor antagonist BQ788 (1 mM) or the NADPH oxidase inhibitor apocynin (1 mM). Reactive oxygen species (ROS) were detected using chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4 0 ,6 0 -diamidino-2 0 -phenylindoladihydrochloride staining and by the caspase-3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl-2, Bax, IkB, caveolin-1 and eNOS was evaluated by Western blot analysis. 2 ET-1 significantly enhanced ROS generation and cell proliferation following 24-h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET-1 to directly upregulate NADPH oxidase. ET-1 itself did not affect apoptosis but attenuated homocysteineinduced apoptosis through an ET B receptor-mediated mechanism. Western blot analysis indicated that ET-1 alleviated homocysteine (Hcy)-induced apoptosis, likely acting by antagonizing the Hcy-induced decreases in Akt, pAkt, pAkt-to-Akt, Bcl-2-to-Bax ratios and increases in Bax and caveolin-1 expression. Furthermore, ET-1 downregulated expression of caveolin-1 and eNOS, which was attenuated by BQ788 or apocynin. 3 In summary, our results suggest that ET-1 affects oxidative stress, proliferation and apoptosis possibly through ET B , NADPH oxidase, Akt, Bax and caveolin-1-mediated mechanisms.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

Inhibition of growth signaling pathways protects against aging and age-related diseases in parallel with reduced oxidative stress. The relationships between growth signaling, oxidative stress and aging remain unclear. Here we report that in Saccharomyces cerevisiae, alterations in growth signaling pathways impact levels of superoxide anions that promote chronological aging and inhibit growth arrest of stationary phase cells in G0/G1. Factors that decrease intracellular superoxide anions in parallel with enhanced longevity and more efficient G0/G1 arrest include genetic inactivation of growth signaling pathways that inhibit Rim15p, which activates oxidative stress responses, and downregulation of these pathways by caloric restriction. Caloric restriction also reduces superoxide anions independently of Rim15p by elevating levels of H2O2, which activates superoxide dismutases. In contrast, high glucose or mutations that activate growth signaling accelerate chronological aging in parallel with increased superoxide anions and reduced efficiency of stationary phase G0/G1 arrest. High glucose also activates DNA damage responses and preferentially kills stationary phase cells that fail to arrest growth in G0/G1. These findings suggest that growth signaling promotes chronological aging in budding yeast by elevating superoxide anions that inhibit quiescence and induce DNA replication stress. A similar mechanism likely contributes to aging and age-related diseases in complex eukaryotes.

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken ␤-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2,7-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4,6-diamidino-2-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0 -200 M) elicited ROS generation and apoptosis in HUVECs in a time-and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehydeinduced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant ␣-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.Chronic alcohol consumption leads to cardiovascular complications such as endothelial dysfunction and alcoholic cardiomyopathy (1). Although several hypotheses have been speculated for alcohol-induced injury including direct/indirect toxicity of alcohol and accumulated fatty acid ethyl esters (2, 3), neither scenario has been fully validated by compelling clinical and experimental evidence. Acetaldehyde is the very first oxidized metabolic product of ethanol and is considered a candidate toxin for alcohol-induced tissue and cell injury. It is far more reactive than ethanol and may inhibit protein synthesis (4, 5). Our laboratory (6 -8) has shown that acetaldehyde interrupts cardiac excitation-contraction coupling and sarco(endo)plasmic reticulum Ca 2ϩ release function. Acetaldehyde has also been shown to form protein adducts leading to atherosclerotic vascular injury (9). Transgenic mice with cardiac overexpression of alcohol dehydrogenase (ADH) 1 displayed higher cardiac acetaldehyde levels associated with compromised heart function at whole heart and ventricular myocyte levels following alcohol intake (10 -12), suggesting that acetaldehyde may be one of t...

Significance We tested the hypothesis that a form of mitochondrial dysfunction alters the homeostasis of the cytosolic Parkinson disease (PD)-associated protein α-synuclein (α-syn). Using yeast and worm models of PD, we show that low levels of phosphatidylethanolamine (PE), caused by the depletion of mitochondrial phosphatidylserine decarboxylase (psd), lead to decreased respiration, endoplasmic reticulum (ER) stress, high levels of α-syn and cytoplasmic α-syn foci, and slow growth. Ethanolamine, which replenishes PE through the Kennedy pathway, diminished ER stress, decreased the level of α-syn, eliminated foci, and restored growth of psd1 Δ cells to near wild-type levels. A low level of mitochondrial PE disrupts the homeostasis of α-syn and leads to the accumulation of cytoplasmic foci of this protein.