PET imaging of in vivo caspase-3/7 activity following myocardial ischemia-reperfusion injury with the radiolabeled isatin sulfonamide analogue [(18)F]WC-4-116.

The utility of [(18)F]WC-4-116, a PET tracer for imaging caspase-3 activation, was evaluated in an animal model of myocardial apoptosis. [(18)F]WC-4-116 was injected into rats at 3 hours after a 30 min period of ischemia induced by temporary occlusion of the left anterior descending coronary artery in Sprague-Dawley rats. [(18)F]WC-4-116 uptake was quantified by 1) autoradiography, 2) microPET imaging studies, and 3) post-PET biodistribution studies. MicroPET imaging also assessed uptake of the non-caspase-3-targeted tracer [(18)F]ICMT-18 at 3 hours postischemia. Enzyme assays and Western blotting assessed caspase-3 activation in both at-risk and not-at-risk regions. Caspase-3 enzyme activity increased in the at-risk but not in the not-at-risk myocardium. Quantitative autoradiographic analysis of [(18)F]WC-4-116 demonstrated nearly 2-fold higher uptake in the ischemia-reperfusion (IR) versus sham animals. [(18)F]WC-4-116 microPET imaging studies demonstrated that the IR animals was similarly elevated in relation to sham. [(18)F]ICMT-18 uptake did not increase in at-risk myocardium despite evidence of caspase-3 activation. Biodistribution studies with [(18)F]WC-4-116 confirmed the microPET findings. These data indicate that the caspase-3-PET tracer [(18)F]WC-4-116 can noninvasively image in vivo caspase activity during myocardial apoptosis and may be useful for clinical imaging in humans.

Long-Term Outcomes in Patients With Diabetes Mellitus Related to Prolonging Clopidogrel More Than 12 Months After Coronary Stenting.

BACKGROUND: Recent large clinical trials show lower rates of late cardiovascular events by extending clopidogrel >12 months after percutaneous coronary revascularization (PCI). However, concerns of increased bleeding have elicited support for limiting prolonged treatment to high-risk patients. OBJECTIVES: The aim of this analysis was to determine the effect of prolonging clopidogrel therapy >12 months versus ≤12 months after PCI on very late outcomes in patients with diabetes mellitus (DM). METHODS: Using the Veterans Health Administration, 28,849 patients undergoing PCI between 2002 and 2006 were categorized into 3 groups: 1) 16,332 without DM; 2) 9,905 with DM treated with oral medications or diet; and 3) 2,612 with DM treated with insulin. Clinical outcomes, stratified by stent type, ≤4 years after PCI were determined from the Veterans Health Administration and Medicare databases and risk was assessed by multivariable and propensity score analyses using a landmark analysis starting 1 year after the index PCI. The primary endpoint of the study was the risk of all-cause death or myocardial infarction (MI). RESULTS: In patients with DM treated with insulin who received drug-eluting stents (DES), prolonged clopidogrel treatment was associated with a decreased risk of death (hazard ratio [HR]: 0.59; 95% confidence interval [CI]: 0.42 to 0.82) and death or MI (HR: 0.67; 95% CI: 0.49 to 0.92). Similarly, in patients with noninsulin-treated DM receiving DES, prolonged clopidogrel treatment was associated with less death (HR: 0.61; 95% CI: 0.48 to 0.77) and death or MI (HR: 0.61; 95% CI: 0.5 to 0.75). Prolonged clopidogrel treatment was not associated with a lower risk in patients without DM or in any group receiving bare-metal stents. CONCLUSIONS: Extending the duration of clopidogrel treatment >12 months may decrease very late death or MI only in patients with DM receiving first-generation DES. Future studies should address this question in patients receiving second-generation DES.

Endovascular intervention for peripheral artery disease.

Advances in endovascular therapies during the past decade have broadened the options for treating peripheral vascular disease percutaneously. Endovascular treatment offers a lower risk alternative to open surgery in many patients with multiple comorbidities. Noninvasive physiological tests and arterial imaging precede an endovascular intervention and help localize the disease and plan the procedure. The timing and need for revascularization are broadly related to the 3 main clinical presentations of claudication, critical limb ischemia, and acute limb ischemia. Many patients with claudication can be treated by exercise and medical therapy. Endovascular procedures are considered when these fail to improve quality of life and function. In contrast, critical limb ischemia and acute limb ischemia threaten the limb and require more urgent revascularization. In general, endovascular treatments have greater long-term durability for aortoiliac disease than femoral popliteal disease. Infrapopliteal revascularization is generally reserved for critical and acute limb ischemia. Balloon angioplasty and stenting are the mainstays of endovascular therapy. New well-tested innovations include drug-eluting stents and drug-coated balloons. Adjunctive devices for crossing chronic total occlusions or debulking plaque with atherectomy are less rigorously studied and have niche roles. Patients receiving endovascular procedures need a structured surveillance plan for follow-up care. This includes intensive treatment of cardiovascular risk factors to prevent myocardial infarction and stroke, which are the main causes of death. Limb surveillance aims to identify restenosis and new disease beyond the intervened segments, both of which may jeopardize patency and lead to recurrent symptoms, functional impairment, or a threatened limb.

Quality of care for patients with acute coronary syndromes as a function of hospital revascularization capability: Insights from get with the guidelines-CAD.

BACKGROUND: Revascularization availability at US hospitals varies and may impact care quality for acute coronary syndrome patients. HYPOTHESIS: The hypothesis of this study was that there would be differences in care quality at Get With The Guidelines-Coronary Artery Disease (GWTG-CAD) hospitals based on revascularization capability. METHODS: For acute coronary syndrome patients admitted to GWTG-CAD hospitals between 2000 and 2010, care quality at hospitals with or without revascularization capability was examined by assessing conformity with performance and quality measures. RESULTS: This study included 95 999 acute coronary syndrome patients admitted to 310 GWTG-CAD hospitals. There were 89 000 patients admitted to 226 revascularization-capable hospitals and 6999 patients admitted to 84 hospitals without revascularization capability included. Adjusted multivariate analysis demonstrated that 8 of the 19 measures were more frequently performed in the revascularization cohort: aspirin (odds ratio [OR]: 1.41, 95% confidence interval [CI]: 1.04-1.92), clopidogrel (OR: 2.31, 95% CI: 1.78-3.00), lipid-lowering therapies at discharge (OR: 1.39, 95% CI: 1.04-1.87), lipid-lowering therapies for low-density lipoprotein >100 mg/dL (OR: 1.85, 95% CI: 1.23-2.77), achievement of blood pressure <140/90 mm Hg (OR: 1.20, 95% CI: 1.03-1.40), LDL recorded (OR: 1.47, 95% CI: 1.05-2.06), and recommendations offered for physical activity (OR: 3.82, 95% CI: 2.23-6.55) or weight management (OR: 1.74, 95% CI: 1.12-2.69). CONCLUSIONS: The GWTG-CAD revascularization hospitals were associated with better performance in some, but not all, measures assessed. Although the difference in conformity between hospital types was modest for performance measures but more variable for quality measures, room for improvement exists in key aspects of care.

Intravascular near-infrared fluorescence molecular imaging of atherosclerosis.

Novel imaging modalities are required to better identify vulnerable atherosclerotic plaques before their dire consequences of myocardial infarction, sudden death, and stroke. Moving beyond traditional diagnostic methods, the field of molecular imaging offers an innovative approach to report upon critical in vivo biological features of high-risk plaques. Molecular imaging employs engineered, targeted imaging agents in conjunction with sophisticated, high-resolution detection systems. While various modalities have been investigated for this purpose, intravascular near infrared fluorescence imaging (NIRF) strategies are uniquely poised to provide high-resolution readouts of human coronary artery plaques. To date, preclinical animal studies have demonstrated feasibility of both standalone NIRF intravascular imaging as well as dual-modality approaches detecting inflammation and fibrin deposition in coronary-sized arteries. This translatable catheter-based approach is positioned to advance the identification of biologically vulnerable coronary plaques and coronary stents at risk of thrombosis.

Neutrophil-mediated accumulation of 2-ClHDA during myocardial infarction: 2-ClHDA-mediated myocardial injury.

The pathophysiological sequelae of myocardial infarction include neutrophil infiltration into the infarct zone that contributes to additional damage to viable tissue and removal of cellular debris from necrosed tissue. Reactive chlorinating species produced by myeloperoxidase amplify the oxidant capacity of activated neutrophils. Plasmalogens are a major phospholipid subclass of both endothelial cells and cardiac myocytes. Recent studies have shown that plasmalogens are targeted by neutrophil-derived reactive chlorinating species that lead to the production of alpha-chloro fatty aldehydes. Results herein demonstrate that the alpha-chloro fatty aldehyde 2-chlorohexadecanal (2-ClHDA) accumulates in rat hearts subjected to left anterior descending coronary artery occlusion. Myocardia from rats subjected to surgical infarction had increased 2-ClHDA and neutrophil infiltration levels compared with myocardia from rats subjected to sham surgery. Additionally, infarcted myocardia from rats rendered neutropenic by treatments with an anti-neutrophil antibody had diminished 2-ClHDA and neutrophil infiltration levels compared with infarcted myocardia from normopenic rats; 2-ClHDA was shown to elicit myocardial damage as determined by lactate dehydrogenase release in isolated perfused rat hearts. Additionally, 2-ClHDA treatment of hearts resulted in decreased heart rate and ventricular performance. Taken together, the present results demonstrate a novel neutrophil-dependent myeloperoxidase-based mechanism that results in 2-ClHDA production in response to regional myocardial infarction that may also contribute to cardiac dysfunction.

Identification of alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions.

BACKGROUND: A role for myeloperoxidase (MPO) as a mediator of coronary artery disease and acute coronary syndromes has recently received considerable attention. Although active MPO and hypochlorite-modified proteins and peptides have been detected in human atherosclerotic lesions, detection of novel chlorinated oxidized lipid species with proatherogenic properties in vivo has not yet been reported. In this study we show that MPO-generated reactive chlorinating species promote selective oxidative cleavage of plasmalogens, liberating alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine in human atherosclerotic lesions. METHODS AND RESULTS: Stable isotope dilution gas chromatography-mass spectrometry methods were used to identify and quantitate the alpha-chloro fatty aldehyde, 2-chlorohexadecanal, in atherosclerotic versus normal human aorta. Compared with normal aorta, 2-chlorohexadecanal levels were elevated more than 1400-fold in atherosclerotic tissues. Parallel electrospray ionization mass spectrometry studies confirmed 34- and 20-fold increases in the plasmalogen cooxidation products, unsaturated lysophosphatidylcholine molecular species containing linoleic and arachidonic acid, respectively, within atherosclerotic compared with normal aorta. Unsaturated lysophosphatidylcholine containing docosahexaenoic acid was also detected in atherosclerotic but not in normal aorta. Exposure of primary human coronary artery endothelial cells to plasmalogen-derived lysophosphatidylcholine molecular species produced marked increases in P-selectin surface expression. CONCLUSIONS: The present studies demonstrate that plasmalogens are attacked by MPO-derived reactive chlorinating species within human atheroma. The resultant species formed, alpha-chloro fatty aldehydes and unsaturated lysophospholipids, possess proatherogenic properties, as shown by induction of P-selectin surface expression in primary human coronary artery endothelial cells.

Characterization of alkylacyl, alk-1-enylacyl and lyso subclasses of glycerophosphocholine by tandem quadrupole mass spectrometry with electrospray ionization.

Positive ion tandem quadrupole mass spectrometric methods for structural characterization of the subclasses of sn-glycero-3-phosphocholine (PC), including alkylacyl- and alk-1-enylacylphosphocholine and lysophosphatidylcholine (LPC), are described. Following collisionally activated dissociation, the [M + Li](+) ions generated by electrospray ionization yield abundant informative fragment ions that permit structural determination, and distinction of regioisomers among lysophosphatidylcholine can be easily achieved. In contrast, structurally informative ions arising from [M + H](+) or [M + Na](+) ions are less prominent. The most abundant ion observed in the product-ion spectra of the [M + Li](+) ions of plasmenyl- and plasmanyl-PC and of LPC arises from loss of N(CH(3))(3) ([M + Li - 59](+)). This feature permits their distinction from a product-ion spectrum arising from a diacylphosphatidylcholine, in which the [M + Li - 183](+) ion reflecting loss of phosphocholine is the most prominent. Examples for identification of various subclasses of PC in biological extracts by tandem mass spectrometry applying various constant neutral loss scannings are also shown.

Myeloperoxidase-derived reactive chlorinating species from human monocytes target plasmalogens in low density lipoprotein.

A role for myeloperoxidase (MPO) in atherosclerosis has received considerable attention recently. To identify potential chlorinated lipid products in human low density lipoprotein (LDL), studies were designed to demonstrate that MPO-derived reactive chlorinating species (RCS) target the plasmalogen pool of LDL isolated from peripheral human blood in vitro. The vinyl ether bond of LDL plasmalogens was targeted by MPO-derived RCS, resulting in the release of the 16- and 18-carbon-containing alpha-chloro fatty aldehydes, 2-chlorohexadecanal and 2-chlorooctadecanal, respectively, from the plasmalogen glycerol backbone. Targeting of the LDL plasmalogen vinyl ether bond was dependent on the presence of MPO-derived RCS. Electrospray ionization mass spectrometric analysis of MPO-treated LDL demonstrated that a novel population of unsaturated lysophosphatidylcholine molecular species was produced by a phospholipase A2-independent mechanism. Unsaturated lysophosphatidylcholine molecular species elicited cyclic AMP response element binding protein phosphorylation in RAW 264.7 cells. Additionally, MPO-mediated targeting of both monocyte and LDL plasmalogen pools was demonstrated in phorbol myristate acetate-stimulated human monocytes, resulting in the production of both 2-chlorohexadecanal and 2-chlorooctadecanal. In contrast, alpha-chloro fatty aldehydes were not produced in phorbol myristate acetate-stimulated mouse monocytes. Collectively, the present studies demonstrate a novel MPO-specific mechanism that mediates the production of a novel group of unsaturated lysophosphatidylcholine molecular species and chlorinated aldehydes from both LDL and monocyte plasmalogen pools that may have important effects during inflammatory reactions mediated by monocytes, most notably atherosclerosis.

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant, alpha-bromo fatty aldehyde.

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.

Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: disparate utilization of sodium halides in the production of alpha-halo fatty aldehydes.

Plasmalogens are a phospholipid molecular subclass that are enriched in the plasma membrane of many mammalian cells. The present study demonstrates that reactive brominating species produced by myeloperoxidase, as well as activated neutrophils, attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens, resulting in the production of a neutral lipid and lysophosphatidylcholine. Gas chromatography-mass spectrometry and proton NMR analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). In comparison to myeloperoxidase-generated reactive chlorinating species, reactive brominating species attacked the plasmalogen vinyl ether bond at neutral pH. In the presence of a 20-fold molar excess of NaCl compared with NaBr, myeloperoxidase-derived reactive halogenating species favored the production of 2-BrHDA over that of 2-chlorohexadecanal. Additionally, 2-BrHDA was preferentially produced from plasmalogen treated with hypochlorous acid in the presence of NaBr. The potential physiological significance of this pathway was suggested by the demonstration that both 2-BrHDA and 2-bromooctadecanal were produced by PMA-stimulated neutrophils. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by myeloperoxidase and by activated neutrophils, resulting in the production of novel brominated fatty aldehydes.

Reactive chlorinating species produced during neutrophil activation target tissue plasmalogens: production of the chemoattractant, 2-chlorohexadecanal.

Recently alpha-chloro fatty aldehydes have been shown to be products of reactive chlorinating species targeting the vinyl ether bond of plasmalogens utilizing a cell-free system. Accordingly, the present experiments were designed to show that alpha-chloro fatty aldehydes are produced by activated neutrophils and to determine their physiologic effects. A sensitive gas chromatography-mass spectrometry technique was developed to detect pentafluorobenzyl oximes of alpha-chloro fatty aldehydes utilizing negative ion chemical ionization. Phorbol 12-myristate 13-acetate activation of neutrophils resulted in the production of both 2-chlorohexadecanal and 2- chlorooctadecanal through a myeloperoxidase-dependent mechanism that likely involved the targeting of both 16 and 18 carbon vinyl ether-linked aliphatic groups present in the sn-1 position of neutrophil plasmalogens. 2-Chlorohexadecanal was also produced by fMLP-treated neutrophils. Additionally, reactive chlorinating species released from activated neutrophils targeted endothelial cell plasmalogens resulting in 2-chlorohexadecanal production. Physiologically relevant concentrations of 2-chlorohexadecanal induced neutrophil chemotaxis in vitro suggesting that alpha-chloro fatty aldehydes may have a role in neutrophil recruitment. Taken together, these studies demonstrate for the first time a novel biochemical mechanism that targets the vinyl ether bond of plasmalogens during neutrophil activation resulting in the production of alpha-chloro fatty aldehydes that may enhance the recruitment of neutrophils to areas of active inflammation.