Cellular mechanisms governing redox homeostasis likely involve their integration with other stresses. Endoplasmic reticulum (ER) stress triggers complex adaptive or proapoptotic signaling defined as the unfolded protein response (UPR), involved in several pathophysiological processes. Since protein folding is highly redox-dependent, convergence between ER stress and oxidative stress has attracted interest. Evidence suggests that ROS production and oxidative stress are not only coincidental to ER stress, but are integral UPR components, being triggered by distinct types of ER stressors and contributing to support proapoptotic, as well as proadaptive UPR signaling. Thus, ROS generation can be upstream or downstream UPR targets and may display a UPR-specific plus a nonspecific component. Enzymatic mechanisms of ROS generation during UPR include: (a) Multiple thiol-disulfide exchanges involving ER oxidoreductases including flavooxidase Ero1 and protein disulfide isomerase (PDI); (b) Mitochondrial electron transport; (c) Nox4 NADPH oxidase complex, particularly Nox4. Understanding the roles of such mechanisms and how they interconnect with the UPR requires more investigation. Integration among such ROS sources may depend on Ca(2+) levels, ROS themselves, and PDI, which associates with NADPH oxidase and regulates its function. Oxidative stress may frequently integrate with a background of ER stress/UPR in several diseases; here we discuss a focus in the vascular system.

Objective-We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis. Methods and Results-We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet ϩ10 4 IU/d Vit.D 2 for 12 weeks. Superoxide and H 2 O 2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H 2 O 2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (PՅ0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA. Conclusions-Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation. Key Words: calcification Ⅲ atherosclerosis Ⅲ antioxidants Ⅲ valves Ⅲ free radicals D egenerative aortic valve (AV) stenosis, the third most prevalent cardiovascular disease in the elderly, 1 shares common risk factors and pathophysiological features with atherosclerosis. [2][3][4][5][6] Although the role of oxidative stress in atherosclerosis is well explored, 7,8 it is unclear whether redox processes contribute to progression of AV calcification. 2,3,9 -11,15,16 Scarce observations provide indirect support for this hypothesis. 10 In vitro studies showed that exogenous superoxide, hydrogen peroxide, or other oxidants increase the number and activity of calcifying vascular cells (CVCs), 11 referred to as a specific subpopulation of cells, derived from (de)differentiation of vascular smooth muscle cells, 12 pericytes, or mesenchymal cells 13 that can produce hydroxyapathite in the vascular wall. 14 In addition, reactive oxygen species (ROS) mediate increase in BMP2 expression and signaling, favoring osteogenesis. 2 On the other hand, calcium resorption by osteoclasts is dependent on ROS derived from its own NAD(P)H oxidase, 15 whereas nitric oxide induces osteoclast detachment and inhibits calcium resorption. 16 Recent data from an experimental mouse model of aortic stenosis suggested locally increased superoxide generation. 3 Observational clinical studies with statins indicated possible decrease in calcification progression in hypercholesterolemic patients, 4 but ...

Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) or PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator.

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