HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acutephase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL 3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

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Intranasal insulin administration raises central nervous system (CNS) insulin levels in humans and acutely facilitates verbal memory in patients with Alzheimer's disease (AD), an effect that may differ by APOE genotype. The purpose of this study was to examine the cognitive dose response curves for intranasal insulin administration, and determine whether the effects of insulin differ between participants with (ε4+) and without (ε4−) the APOE-ε4 allele. On separate mornings, 33 memoryimpaired adults with AD or amnestic mild cognitive impairment and 59 normal adults each underwent five intranasal treatment conditions consisting of insulin (10, 20, 40, or 60 IU) or placebo. Cognition was tested 15-minutes post-treatment, and blood was acquired at baseline and 45-minutes posttreatment. Plasma insulin and glucose levels were unaffected by treatment. Insulin administration facilitated recall on two measures of verbal memory in memory-impaired ε4− adults, with performance generally peaking at 20 IU. In contrast, memory-impaired ε4+ subjects demonstrated a relative decline in verbal memory. Insulin also differentially modulated plasma β-amyloid for memory-impaired subjects and normal controls, effects that again differed by APOE genotype. These findings suggest that groups with different genetic risks for AD may show differential dose-response curves following intranasal insulin administration.

Epidemiological studies associate post-menopausal estrogen use with a reduction in risk of Alzheimer's disease, a reduction in risk of Parkinson's disease, and death from stroke. The neuroprotective efficacy of estrogens have been well described and may contribute to these clinical effects. Estrogen-mediated neuroprotection has been described in several neuronal culture model systems with toxicities including serum-deprivation, beta-amyloid-induced toxicity, excitotoxicity, and oxidative stress. In animal models, estrogens have been shown to attenuate neuronal death in rodent models of cerebral ischemia, traumatic injury, and Parkinson's disease. Although estrogens are known to exert several direct effects on neurons, the cellular mechanisms behind the neuroprotective efficacy of the steroid are only beginning to be elucidated. In this review, we summarize the data supporting a neuroprotective role for estrogens in both culture and animal models and discuss neuronal effects of estrogens that may contribute to the neuroprotective effects. These effects include activation of the nuclear estrogen receptor, altered expression of bcl-2 and related proteins, activation of the mitogen activated kinase pathway, activation of cAMP signal transduction pathways, modulation of intracellular calcium homeostasis, and direct antioxidant activity.

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High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but itis not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 ؎ 178 mol/mol Tyr) than that in circulating HDL (104 ؎ 11 mol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, proteinbound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo. High density lipoprotein (HDL)3 protects against atherosclerosis by removing cholesterol from cells of the artery wall (1, 2). Apolipoprotein A-I (apoA-I), which accounts for ϳ70% of the total protein in HDL, promotes cholesterol and phospholipid efflux largely by an active transport process mediated by ATP-binding cassette transporter A1 (ABCA1). However, oxidation of apoA-I severely impairs cholesterol efflux by the ABCA1 pathway (3-5). Moreover, HDL is chlorinated in human atherosclerotic lesions and the blood of subjects with established coronary artery disease, suggesting that apoA-I oxidation might promote atherogenesis.One potential pathway for apoA-I oxidation involves myeloperoxidase (MPO), a heme protein expressed by macrophages in human atherosclerotic tissue (6 -8). MPO secreted by phagocytes uses hydrogen peroxide (H 2 O 2 ) and chloride (Cl Ϫ ) to generate the powerful oxidant hypochlorous acid (HOCl). HOCl converts tyrosine to 3-chlorotyrosine (8 -10), and MPO is the only known source of this halogenated amino acid during acute inflammation in mice (11), indicating that MPO oxidizes HDL in vivo. We previously showed that MPO or HOCl targets tyrosine residue 192 (Tyr-192) when it chlorinates apoA-I, regardless of whether the protein is free or associated with HDL (12, 13). Moreover, apoA-I loses its ability to remove cholesterol from cells as it becomes oxidized in this manner (3-5, 13), i...