Sean S. Davies

Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in mice and humans.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited arrhythmia syndrome in which drug therapy is often ineffective. We discovered that flecainide prevents arrhythmias in a mouse model of CPVT by inhibiting cardiac ryanodine receptor–mediated Ca2+ release and thereby directly targeting the underlying molecular defect. Flecainide completely prevented CPVT in two human subjects who had remained highly symptomatic on conventional drug therapy, indicating that this currently available drug is a promising mechanism-based therapy for CPVT.

Inflammatory Platelet-activating Factor-like Phospholipids in Oxidized Low Density Lipoproteins Are Fragmented Alkyl Phosphatidylcholines

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A1 greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C4-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C4-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C4-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C4-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.

Isoprostane Generation and Function

Free radicals derived primarily from molecular oxygen have been implicated in a variety of human disorders including atherosclerosis, cancer, neurodegenerative diseases, and aging.1 Damage to tissue biomolecules, including lipids, proteins, and DNA, by free radicals is postulated to contribute importantly to the pathophysiology of oxidative stress. Lipids are readily attacked by free radicals resulting in the formation of a number of peroxidation products.2 The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via the non-enzymatic free radical-initiated peroxidation of arachidonic acid, a ubiquitous polyunsaturated fatty acid (PUFA). Since discovery of these molecules over twenty years ago by Morrow and Roberts, one class of IsoPs, the F2-IsoPs, have become the biomarker of choice for assessing endogenous oxidative stress because these molecules are chemically stability and have been detected in all biological fluids and tissues analyzed.3-5 In addition to F2-IsoPs, a variety of IsoPs with different ring structures have been identified. Several of these compounds possess potent biological activities that could account for some of the pathophysiological effects of oxidative injury. Further, IsoP-like molecules are also generated from a number of different PUFAs including α-linolenic acid, eicosapentaenoic acid (EPA), adrenic acid, and docosahexaenoic acid (DHA) (Figure 1). There are many excellent reviews in the literature describing not only the quantification of F2-IsoPs in human health and disease but also the biological activities of these molecules.6-9 Thus, this review seeks to give readers a comprehensive, up-to-date overview of our current knowledge regarding IsoPs including the chemistry and biochemistry of their formation and metabolism, the utility of measuring these compounds as markers of in vivo oxidant stress, and their biological properties.

DC isoketal-modified proteins activate T cells and promote hypertension

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1β, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

Oxidative Mediated Lipid Peroxidation Recapitulates Proarrhythmic Effects on Cardiac Sodium Channels

Sudden cardiac death attributable to ventricular tachycardia/fibrillation (VF) remains a catastrophic outcome of myocardial ischemia and infarction. At the same time, conventional antagonist drugs targeting ion channels have yielded poor survival benefits. Although pharmacological and genetic models suggest an association between sodium (Na+) channel loss-of-function and sudden cardiac death, molecular mechanisms have not been identified that convincingly link ischemia to Na+ channel dysfunction and ventricular arrhythmias. Because ischemia can evoke the generation of reactive oxygen species, we explored the effect of oxidative stress on Na+ channel function. We show here that oxidative stress reduces Na+ channel availability. Both the general oxidant tert-butyl-hydroperoxide and a specific, highly reactive product of the isoprostane pathway of lipid peroxidation, E2-isoketal, potentiate inactivation of cardiac Na+ channels in human embryonic kidney (HEK)-293 cells and cultured atrial (HL-1) myocytes. Furthermore, E2-isoketals were generated in the epicardial border zone of the canine healing infarct, an arrhythmogenic focus where Na+ channels exhibit similar inactivation defects. In addition, we show synergistic functional effects of flecainide, a proarrhythmic Na+ channel blocker, and oxidative stress. These data suggest Na+ channel dysfunction evoked by lipid peroxidation is a candidate mechanism for ischemia-related conduction abnormalities and arrhythmias.

Clinical Relevance of Biomarkers of Oxidative Stress

Abstract Significance: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. Recent Advances: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. Critical Issues: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. Future Directions: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker. Antioxid. Redox Signal. 23, 1144–1170.

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a persons microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

F2-isoprostanes as an indicator and risk factor for coronary heart disease

Coronary heart disease (CHD) is the leading single cause of death in the United States and most Western countries, killing more than 400,000 Americans per year. Although CHD often manifests suddenly as a fatal myocardial infarction, the atherosclerosis that gives rise to the infarction develops gradually and can be markedly slowed or even reversed through pharmacological and lifestyle interventions. These same atherosclerotic processes also drive related vascular diseases such as stroke and peripheral artery disease, and individuals surviving occlusive events often develop additional complications including ischemic cardiomyopathy and heart failure. Therefore, better detection of subclinical atherosclerosis, along with more effective treatments, could significantly reduce the rate of death from CHD and related vascular diseases in the United States. In recent years, oxidation of polyunsaturated fatty acids (PUFAs) in plasma lipoproteins has been postulated to be a critical step in the development of atherosclerosis. If so, then monitoring lipid peroxidation should be a useful indicator of disease risk and progression. This review focuses on the evidence that specific PUFA peroxidation products, the F(2)-isoprostanes, are useful biomarkers that could potentially be utilized as indicators of CHD.

The Biochemistry of the Isoprostane, Neuroprostane, and Isofuran Pathways of Lipid Peroxidation

Isoprostanes are prostaglandin‐like compounds that are formed non‐enzymatically by free radical‐catalyzed peroxidation of arachidonic acid (C20:4ω6). Intermediates in the pathway of the formation of isoprostanes are labile prostaglandin H2‐like bicyclic endoperoxides (H2‐isoprostanes). H2‐isoprostanes are reduced to form F‐ring isoprostanes (F2‐isoprostanes), but they also undergo chemical rearrangement in vivo to form E2‐ and D2‐isoprostanes, isothromboxanes, and highly reactive acyclic γ‐ketoaldehdyes (isoketals). E2‐ and D2‐isoprostanes also undergo dehydration in vivo to form cyclopentenone A2‐ and J2‐isoprostanes. Docosahexaenoic acid (C22:6ω3) is highly enriched in neurons in the brain and is highly susceptible to oxidation. Free radical‐catalyzed oxidation of docosahexaenoic acid results in the formation of isoprostane‐like compounds (neuroprostanes). F4‐,D4‐,E4‐,A4‐,and J4‐neuroprostanes and neuroketals have all been shown to be produced in vivo. In addition, we recently discovered a new pathway of lipid peroxidation that forms compounds with a substituted tetrahydrofuran ring (isofurans). Oxygen concentration differentially modulates the formation of isoprostanes and isofurans. As oxygen concentrations increase, the formation of isofurans is favored whereas the formation of isoprostanes becomes disfavored.

Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function.

Oxidative stress can impair proteasome function, both of which are features of neurodegenerative diseases. Inhibition of proteasome function leads to protein accumulation and cell death. We discovered recently the formation of highly reactive γ‐ketoaldehydes, isoketals (IsoKs), and neuroketals (NeuroKs) as products of the isoprostane and neuroprostane pathways of free radical‐induced lipid peroxidation that are analogous to cyclooxygenase‐derived levuglandins (LGs). Because aldehydes that are much less reactive than IsoKs have been shown to inhibit proteasome function, we explored the ability of the proteasome to degrade IsoK‐adducted proteins/peptides and the effect of IsoK and IsoK‐adducted proteins/peptides on proteasome function. Adduction of IsoK to model proteasome substrates significantly reduced their rate of degradation by the 20S proteasome. The ability of IsoK to inhibit proteasome function directly was observed only at very high concentrations. However, at much lower concentrations, an IsoK‐adducted protein (ovalbumin) and peptide (Aβ1–40) significantly inhibited chymotrypsin‐like activity of the 20S proteasome. Moreover, incubation of IsoK with P19 neuroglial cultures dose‐dependently inhibited proteasome activity (IC50 = 330 nM) and induced cell death (LC50 = 670 nM). These findings suggest that IsoKs/NeuroKs/LGs can inhibit proteasome activity and, if overproduced, may have relevance to the pathogenesis of neurodegenerative diseases.