Objective Diminished cholesterol efflux activity of apolipoprotein B (apoB)–depleted serum is associated with prevalent coronary artery disease, but its prognostic value for incident cardiovascular events is unclear. We investigated the relationship of cholesterol efflux activity with both prevalent coronary artery disease and incident development of major adverse cardiovascular events (death, myocardial infarction, or stroke). Approach and Results Cholesterol efflux activity from free cholesterol–enriched macrophages was measured in 2 case– control cohorts: (1) an angiographic cohort (n=1150) comprising stable subjects undergoing elective diagnostic coronary angiography and (2) an outpatient cohort (n=577). Analysis of media from cholesterol efflux assays revealed that the high-density lipoprotein fraction (1.063

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 µM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 µM), mid (5 µM) and high (100 µM) levels of 98.2%, 97.3% and 101.6%, respectively. Additional assay performance metrics include intra-day and inter-day coefficients of variance of < 6.4% and < 9.9%, respectively, across the range of TMAO levels. Stability studies reveal TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) shows a range of 0.73 – 126 µM, median (interquartile range) levels of 3.45 (2.25–5.79) µM, and increasing values with age. The LC/MS/MS based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic and environmental factors on TMAO levels.

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr 71 ), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

An essential feature of the innate immune system is maintaining cellular homeostasis by identifying and removing senescent and apoptotic cells and modified lipoproteins. Identification is achieved through the recognition of molecular patterns, including structurally distinct oxidized phospholipids, on target cells by macrophage receptors. Both the structural nature of the molecular patterns recognized and their orientation within membranes has remained elusive. We recently described the membrane conformation of an endogenous oxidized phospholipid ligand for macrophage scavenger receptor CD36, where the truncated oxidized sn-2 fatty acid moiety protrudes into the aqueous phase, rendering it accessible for recognition. Herein we examine the generality of this conformational motif for peroxidized glycerophospholipids within membranes. Our data reveal that the addition of a polar oxygen atom on numerous peroxidized fatty acids reorients the acyl chain, whereby it no longer remains buried within the membrane interior but rather protrudes into the aqueous compartment. Moreover, we show that neither a conformational change in the head group relative to the membrane surface nor the presence of a polar head group is essential for CD36 recognition of free oxidized phospholipid ligands within membranes. Rather, our results suggest the following global phenomenon. As cellular membranes undergo lipid peroxidation, such as during senescence or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of the lipid bilayer to the aqueous exterior. This enables physical contact between pattern recognition receptor and molecular pattern ligand. Cell membranes thus "grow whiskers" as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface.The fluid mosaic model of membrane architecture, first proposed by Singer and Nicolson (1), is a cornerstone of cell biology. An integral feature of this model is the macromolecular assembly of amphipathic phospholipids into a bilayer structure, with polar head groups directed toward the aqueous phase and hydrophobic aliphatic fatty acid chains of glycerophospholipids extending toward the membrane interior. The bilayer structure posited explains how a cell membrane maintains a critical barrier function while it simultaneously facilitates rapid lateral diffusion of proteins and lipids within the planar membrane surface.Some biological processes, however, are not compatible with a classic phospholipid orientation as described for a lamellar (bilayer) phase membrane structure and instead involve alternative phospholipid conformations. For example, membrane fusion of two vesicles must be accompanied by a transient nonlamellar phase orientation of the phospholipids at the site of membrane fusion (2, 3). Perhaps less obvious is the recognition of a phospholipid ligand within a membrane bilayer of one cell by an alternative cell's receptor. It is hard to envision how a macrophage pattern recognition receptor like CD36 can identify se...

The predicted structure and molecular trajectories from over 80 ns molecular dynamics simulation of the solvated double super helix (DSH) model of nascent-high density lipoprotein (HDL) was determined and compared with experimental data on reconstituted nascent HDL obtained from multiple biophysical platforms including small angle neutron scattering (SANS) with contrast variation, hydrogen-deuterium exchange tandem mass spectrometry (H/D-MS/MS), nuclear magnetic resonance spectroscopy (NMR), cross-linking tandem mass spectrometry (MS/MS), fluorescent resonance energy transfer (FRET), electron spin resonance spectroscopy (ESR), and electron microscopy. In general, biophysical constraints experimentally derived from the multiple platforms agree with the same quantities evaluated using the simulation trajectory. Notably, key structural features postulated for the recent DSH model of nascent HDL are retained during the simulation including: 1) the super helical conformation of the anti-parallel apolipoprotein A1 (apoA1) chains; 2) the lipid micellar-pseudolamellar organization; and 3) the solvent exposed Solar Flare loops, proposed sites of interaction with LCAT (lecithin cholesteryl acyltransferase). Analysis of salt bridge persistence during simulation provides insights into structural features of apoA1 that form the backbone of the lipoprotein. The combination of molecular dynamics simulation and experimental data from a broad range of biophysical platforms serves as a powerful approach to study large macromolecular assemblies such as lipoproteins. The present application to nascent HDL validates the DSH model proposed earlier, and suggests new structural details of nascent HDL. 4 To whom correspondence should be addressed: Cleveland State University, 2121 Euclid Avenue, SI 422, Cleveland, OH 44115, or Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, v.gogonea@csuohio.edu or hazens@ccf.org,. SUPPORTING INFORMATION AVAILABLE. The supporting information contains Suplemmental Figures 1 and 2 that depict small angle neutron scattering intensities (I(q)) versus the scattering vector (q), and distance distribution functions (p(r)) versus the distance between scattering centers (r), and low-resolution structures of nascent rHDL in 12% and 42% D 2 O. The Supplemental Table 1 lists experimental and calculated per residue deuterium incorporation factors, H/D exchange rate constants, and residue unfolding constants. This material is available free of charge via the Internet at http://pubs.acs.org. Additionally, the following information is available for download from http://www.lerner.ccf.org/cellbio/hazen/data/ -neutron scattering intensities for HDL samples analyzed in 12% and 42% D 2 O (text files), low resolution structures of nascent HDL (pdb files), and calculated H/D exchange deuterium incorporation factors for all residues in apoA1 dimer (Excel file). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 August 31. NIH-PA Author ManuscriptNIH-PA Author Manuscri...

Phagocytic removal of aged or oxidatively damaged cells and macromolecules is an indispensable homeostatic function of the innate immune system. A structurally conserved family of oxidized phospholipids that serve as endogenous high-affinity ligands for the macrophage scavenger receptor CD36 (oxPC(CD36)) was recently identified. Enriched within atherosclerotic plaque and senescent cell membranes, oxPC(CD36) promote the uptake of oxidized lipoproteins and cell membranes by macrophages when present at only a few molecules per particle. How macrophages recognize oxPC(CD36) within cellular membranes and lipoprotein surfaces remains unknown. Herein, we deduce the conformation of oxPC(CD36) near the hydrophobic-hydrophilic interface within membrane bilayers by determining multiple critical internuclear distances using nuclear Overhauser enhancement spectroscopy. The molecular model reveals a unique conformation for oxPC(CD36) within bilayers whereby the distal end of the sn-2 acyl chain harboring the structurally conserved CD36 recognition motif protrudes into the aqueous phase. The remarkable conformation elucidated for oxPC(CD36) produces a surface accessible phagocytic "eat me signal" to facilitate senescent cell and oxidized lipoprotein recognition by the scavenger receptor CD36 as part of its immune surveillance function.

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Between April 1997 and February 2000, total cavopulmonary connection with an extraatrial tunnel was used to treat 9 cases of complicated congenital heart disease: single ventricle (4), double-outlet right ventricle (3), mitral atresia (1), and tricuspid atresia (1). There was no mortality. One patient developed bacterial endocarditis and required reoperation after 52 days to replace the tunnel. At follow-up ranging from 11 months to 3 years, 3 patients were in New York Heart Association functional class I, and 6 were in class II. One patient with single ventricle had refractory supraventricular tachycardia after a modified Fontan operation 4 years earlier, which was cured by the total cavopulmonary connection procedure. The essential factors for a good outcome include appropriate surgical indication, avoidance of aortic crossclamping and cardiac arrest, and unobstructed anastomosis between the superior and inferior venae cavae and the pulmonary artery.