John F. Keaney

Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance

Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (rho(0) cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.

Acute EGCG Supplementation Reverses Endothelial Dysfunction in Patients with Coronary Artery Disease

Background: Epidemiological studies demonstrate an inverse relation between dietary flavonoid intake and cardiovascular risk. Recent studies with flavonoid-containing beverages suggest that the benefits of these nutrients may relate, in part, to improved endothelial function. Objective: We hypothesized that dietary supplementation with epigallocatechin gallate (EGCG), a major catechin in tea, would improve endothelial function in humans. Design: We examined the effects of EGCG on endothelial function in a double blind, placebo-controlled, crossover design study. We measured brachial artery flow-mediated dilation by vascular ultrasound at six time points: prior to treatment with EGCG or placebo, two hours after an initial dose of EGCG (300 mg) or placebo, and after two weeks of treatment with EGCG (150 mg twice daily) or placebo. The order of treatments (EGCG or placebo) was randomized and there was a one-week washout period between treatments. Results: A total of 42 subjects completed the study, and brachial artery flow-mediated dilation improved from 7.1 ± 4.1 to 8.6 ± 4.7% two hours after the first dose of 300 mg of EGCG (P = 0.01), but was similar to baseline (7.8 ± 4.2%, P = 0.12) after two weeks of treatment with the final measurements made approximately 14 hours after the last dose. Placebo treatment had no significant effect, and there were no changes in reactive hyperemia or the response to sublingual nitroglycerin. The changes in vascular function paralleled plasma EGCG concentrations, which increased from 2.6 ± 10.9 to 92.8 ± 78.7 ng/ml after acute EGCG (P < 0.001), but were unchanged from baseline after two weeks of treatment (3.4 ± 13.1 ng/ml). Conclusion: EGCG acutely improves endothelial function in humans with coronary artery disease, and may account for a portion of the beneficial effects of flavonoid-rich food on endothelial function.

Vitamin E and vascular homeostasis: implications for atherosclerosis

Considerable epidemiologic data suggest that dietary consumption of vitamin E reduces the incidence of cardiovascular disease. The precise mechanisms are not clear, but emerging data indicate that vitamin E has numerous activities that may, in part, explain its effect on vascular disease. In particular, vitamin E enhances the bioactivity of nitric oxide, inhibits smooth muscle proliferation, and limits platelet aggregation. One common mechanism to account for these effects of vitamin E is the inhibition of protein kinase C stimulation. In the setting of atherosclerosis, inhibition of protein kinase C by vitamin E would be expected to maintain normal vascular homeostasis and thus reduce the clinical incidence of cardiovascular disease.—Keaney, J. F., Jr., Simon, D. I., Freedman, J. E. Vitamin E and vascular homeostasis: implications for atherosclerosis. FASEB J. 13, 965–976 (1999)

Oxidative stress, antioxidants, and endothelial function.

Endothelial dysfunction, characterized by a loss in nitric oxide bioactivity, is an early event in the development of atherosclerosis and determines future vascular complications. Emerging evidence suggests a causal role for oxidative stress in this process. Reactive oxygen species can directly inactivate nitric oxide, modulate protein function and act as cellular signaling molecules. These events contribute to the initiation and progression of endothelial dysfunction. Considerable data also indicates that antioxidant compounds limit oxidative damage and restore endothelial function. The purpose of this review is to discuss these data and suggest novel approaches for lowering the oxidative stress in the vasculature.

Atherosclerosis: from lesion formation to plaque activation and endothelial dysfunction.

Atherosclerosis is an important source of morbidity and mortality in the developed world. Despite the fact that the association between LDL cholesterol and atherosclerosis has been evident for at least three decades, our understanding of exactly how LDL precipitates atherosclerosis is still in its infancy. At least three working hypotheses of atherosclerosis are now nearing the stage where their critical evaluation is possible through a combination of basic science investigation and murine models of atherosclerosis. As we move forward in our understanding of this disease, efforts will be increasingly focused on the molecular mechanisms of disease activation that precipitate the clinical manifestations of atherosclerosis such as heart attack and stroke. Two candidates for such investigation involve the events surrounding plaque activation and endothelial dysfunction. Further investigation in these fields should provide the necessary insight to develop the next generation of interventions that will reduce the clinical manifestations of this devastating disease. The purpose of this work is to review the major theories of atherogenesis, examine the aspects of atherosclerosis that lead to disease activation and discuss aspects of disease activation that are amenable to treatment.

Reactive oxygen species-mediated signal transduction in the endothelium.

The endothelium is an important component of vascular homeostasis that is a target for injury in the setting of vascular disease. One means of promoting a maladaptive endothelial cell phenotype such as that seen in atherosclerosis is excess oxidative stress. Although this term once was almost exclusively used to describe low-density lipoprotein (LDL) and lipid oxidation in the vasculature, we now understand that the intracellular oxidant milieu is an important modulator of vascular cell function. Indeed, considerable data indicate that reactive oxygen species (ROS) are an important means of cellular signaling, although the precise mechanisms whereby ROS accomplish this are still under investigation. In this review, the data linking ROS to kinase activation and cell signaling in the endothelium is discussed, with a particular emphasis on the roles of protein thiol modification.

NITRIC OXIDE AND SUPEROXIDE DETECTION IN HUMAN PLATELETS

Publisher Summary This chapter discusses the nitric oxide and superoxide detection in human platelets. Activation and recruitment of platelets is tightly regulated. Adhesion of platelets to normal endothelium is prevented by several mechanisms, including endothelial cell production of prostacyclin and nitric oxide (NO . ). Nitric oxide inhibits platelet aggregation and prevents thrombosis in a model of endotoxins-induced glomerular damage. A constitutive nitric oxide synthase (cNOS) has been identified in both human platelets and megakaryoblastic cells. Platelets incubated with cNOS inhibitors demonstrate enhanced aggregation, whereas the cNOS substrate L-arginine inhibits aggregation. Platelets also produce superoxide (O 2 - ) during aggregation. Superoxide and NO- react readily, producing OONO-. It is possible that platelet O 2 - production modifies the bioavailability of NO . for platelets. For this reason, it is relevant to define both NO . and O 2 -release from platelets. The chapter also discusses NO-selective microelectrode for use in a standard platelet aggregometer.

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.

Preparation of lipid hydroperoxide-free low-density lipoproteins

Publisher Summary Lipid hydroperoxides formed during low density lipoproteins (LDL) isolation are important determinants of subsequent LDL resistance to metal ion-dependent oxidation and can mask or alter effects of LDL antioxidant manipulation. Thus, of paramount importance in studying the oxidation of LDL ex vivo is the prevention of adventitious oxidation during its isolation and handling in preparation for such assays. This chapter describes a method for isolating LDL in a manner that prevents artifactual oxidation, yielding an LDL preparation containing the same amounts of lipid hydroperoxides, if any, as LDL in plasma. Modifications of the method described by Redgrave have been used combining standard fixed angle or swinging bucket rotors with one or two centrifugations of 20 h or more. More recently, with the advent of near-vertical and vertical rotors, simple two-step gradients have been employed with durations of ultracentrifugation as brief as 25 min for isolation of LDL. These methods described by Chung and colleagues have several advantages, including significantly shorter centrifugation times and ease of preparation with underlaying of density adjusted plasma in a simple two-step gradient; the methods also minimize apoprotein redistribution and oxidation of lipoproteins that may occur during longer periods of ultracentrifugation.

Determination of phospholipid oxidation in cultured cells.

Publisher Summary This chapter discusses the use of high-performance liquid chromatography (HPLC) with postcolumn chemiluminescence detection for the measurement of lipid peroxidation in cultured cells. In this assay, extracted phospholipids are separated by HPLC over a silica column and individual lipid classes are detected by UV (ultra violet) absorption. In the postcolumn chemiluminescence assay, microperoxidase catalyzes the decomposition of hydroperoxides to the corresponding alkoxyl radicals. This alkoxyl radical formation facilitates the oxidation and decomposition of isoluminol, yielding visible light that is maximal at a wavelength of 430 nm. This method has several advantages over the more common thiobarbituric acid (TBA) assay. Specifically, this assay measures the hydroperoxide groups directly, and it is less prone to interference such as is observed with the TBA assay. In addition, the assay can be adapted for individual identification of phospholipid hydroperoxides (PLOOHs) from the different phospholipid classes.