Published online before print July 5, 2007, doi: 10.1161/CIRCRESAHA.107.157537
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Increased Protein Nitration Burden in the Atherosclerotic Lesions and Plasma of Apolipoprotein A-I–Deficient Mice
From the Stokes Research Institute and Departments of Pediatrics and Pharmacology (I.P., L.T., D.M.F., H.I.), Children’s Hospital of Philadelphia and The University of Pennsylvania, Philadelphia; the Department of Medicine (R.E.M., J.T., D.J.R.), The University of Pennsylvania, Philadelphia; the Department of Cardiovascular Medicine and Center for Cardiovascular Diagnostics and Prevention (X.F., S.L.H.), Cleveland Clinic Foundation, Cleveland Ohio; the Laboratory of Clinical Chemistry and Hematology and Cell Microscopy Center (H.F.G.H.), University Medical Center Utrecht, and Institute for Biomembranes, Utrecht, The Netherlands; the Department of Biochemistry and Mass Spectrometry Research Center (M.K.D., D.C.L.), Vanderbilt University School of Medicine, Nashville, Tenn; and the Department of Biological Chemistry (I.P.), School of Medicine, Aristotle University of Thessaloniki, Greece. Permanent address for L.T.: Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Current address for D.M.F.: Ministry of Health, Sao Paulo, SP, Brazil.
Correspondence to Harry Ischiropoulos, Stokes Research Institute, Children’s Hospital of Philadelphia, 416D Abramson Research Center, 34th Street Civic Center Boulevard, Philadelphia, PA 19104-4318. E-mail ischirop{at}mail.med.upenn.edu
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Abstract |
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Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA–apoA-I–/–). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA–apoA-I–/– as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA–apoA-I–/– as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA–apoA-I–/– that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA–apoA-I–/– as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.
Key Words: atherosclerosis • tyrosine nitration • proteomics
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Introduction |
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The levels of high-density lipoprotein (HDL) in plasma are inversely related to the development of atherosclerosis. The major protein component in HDL is apolipoprotein A-I (apoA-I). We previously reported that low-density lipoprotein receptor (LDLr) and apobec-1 null double-knockout mice lacking apoA-I (LA–apoA-I–/–) had more extensive atherosclerotic lesions than LDLr/apobec-1 knockout mice (LA) with normal apoA-I despite the well-preserved levels of HDL-cholesterol1 (HDL-C), confirming the specific importance of apoA-I in inhibiting atherogenesis. In vitro, apoA-I has a variety of properties that could be antiatherogenic, including promotion of macrophage cholesterol efflux,2 inhibition of LDL oxidation,3 reduction of lipid hydroperoxides,4 inhibition of the endothelial inflammatory response,5,6 and promotion of endothelial nitric oxide production.7–9 Interestingly, apoA-I is modified by tyrosine nitration and chlorination in both plasma and artery walls and the extent of these modifications is significantly higher in coronary artery disease patients (CAD) compared with healthy individuals.10 The chlorination and nitration of tyrosine residues in apoA-I are facilitated by its close association with myeloperoxidase (MPO), a source of nitrating and chlorinating oxidants, as shown previously by immunohistochemistry and coimmunoprecipitation experiments.10,11 We hypothesized that apoA-I modulates the oxidative burden during the development of atherosclerosis. To test this hypothesis, proteins modified by nitrating oxidants were quantified and localized in atherosclerotic lesions in atherosclerosis-prone mice lacking apoA-I and compared with those expressing wild-type apoA-I. Furthermore a proteomic survey of nitrated proteins in the plasma was performed to identify the predominant protein targets for nitrating oxidants that could potentially serve as biomarkers. Collectively the data revealed that the lack of apoA-I increased the oxidant-modified protein burden in these atherosclerosis-prone mice.
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Materials and Methods |
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Biological Samples
Aortic roots and plasma were obtained from 10-month-old female C57BL6/J (wild-type), LDLr–/–, apobec1–/– double knockout (LA), and LDLr–/–, apobec1–/–, apoA-I–/– triple knockout (LA–apoA-I–/–) as described previously.1,12
Immunohistochemistry, electron microscopy, and Western blotting were performed as described previously.13–15 An expanded description of the immunohistochemical and Western blotting methods can be found in the supplemental material (available online at http://circres.ahajournals.org).
Quantification of Aortic Root Protein Nitrotyrosine Levels
The levels of protein nitrotyrosine were quantified aortic roots by high performance liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) using stable isotope dilution methodology as described previously.10
Enrichment of Nitrated Proteins and Protein Identification by MS/MS
Proteins modified by nitration in the mice plasma were enriched by affinity capture using solid phase cross linked affinity purified polyclonal antinitrotyrosine antibodies. The detailed methods for the affinity enrichment and identification of proteins my MS/MS are described in the supplemental material.
Evaluating Sequest Peptide Sequence Assignments
Peptide sequences matched to MS/MS spectra by Sequest and corresponding protein identifications were accepted based on the following selection criteria. First, the protein must be identified in both replicate experiments, with at least 2 distinct peptides matching the following Sequest Xcorr (XC) scores: XC>2.5 for doubly charged and >3.5 for triply charged ions; 
Cn >0.1; RSp <5; and preliminary score (Sp) >350. These criteria resulted in false-positive identification rates of 0 to 3.2%, which were estimated from reversed-sequence identifications in the database. Assigned spectra that met the above criteria were then manually reviewed. For peptide assignments to be accepted they must have (1) a continuous b or y-ion series of at least 5 residues and (2) the top 3 most intense fragment peaks assigned to either an a, b, y-ion, to an a, b, y-ion resulting from a neutral loss of water or ammonia, or to a multiply protonated fragment ion.
Ligand Competition ELISA
Antinitrotyrosine antibody detection was performed as described in detail previously.16
Fibrinogen Isolation, Western Blotting, and Polymerization Assays
Fibrinogen was isolated from human plasma by glycine precipitation. Fibrinogen concentration was determined by BCA protein assay (Pierce) using purified fibrinogen from American Diagnostica as the standard. Nitrated molecules removal was performed by immunoprecipitation. Briefly, 200 µg of antinitrotyrosine antibodies were covalently attached to 400-µL amino-link plus agarose beads (Pierce Biotechnology). Two hundred fifty micrograms of fibrinogen was loaded to the beads and incubated overnight rotating end-over-end at 4°C. The beads were then centrifuged at 3000g for 1 minute, and the unbound fractions (3-nitrotyrosine depleted) were collected. The beads were washed with 10 volumes TBS, then with 6 volumes 0.5 mol/L NaCl, and finally with 10 volumes TBS. The captured fractions were eluted with 4 volumes 50 mmol/L glycine pH 2.8, neutralized with 1 mol/L Tris pH 9 and concentrated to a small volume (10 to 20 µL) using YM-10 µm filters (Millipore). For the control experiments, 200 µg of isolated fibrinogen were loaded in bovine IgG-coupled beads and underwent the same process as described above. The unbound fractions of these columns will be referred as the nondepleted fibrinogen.
Western blotting for fibrinogen and 3-nitrotyrosine was performed using a polyclonal anti-human fibrinogen antibody (#A0080, DAKO) and the polyclonal antinitrotyrosine antibody 609.13,15
For fibrinogen clotting assays, 3-nitrotyrosine depleted and nondepleted samples were adjusted to 0.4 mg/mL with TBS and polymerization was initiated by the simultaneous addition of human 
-thrombin (American Diagnostica) and CaCl2 to a final concentration of 1 NIH U/mL and 5 mmol/L, respectively. Turbidity changes were monitored at 350 nm using a plate reader (Spectramax 250, Molecular Devices).
Statistical Analysis
Values are presented as mean±SD. Statistical significance was determined by student t test using SigmaStat v2.03.
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Results |
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Immunohistochemical Detection of Protein Nitration in Aortic Lesions
Affinity purified antinitrotyrosine antibodies, which have been previously characterized in detail,15 were used to detect nitrated proteins in aortic root sections from 10-month-old LA and LA–apoA-I–/– mice. Staining was observed primarily within the atherosclerotic lesions, and it was more robust in LA apoA-I–/– (Figure 1A, right panel) as compared with LA lesions (Figure 1A, left panel). To ascertain the specificity of staining, the antinitrotyrosine antibodies were competed with the synthetic octapeptide used to generate these antibodies. Competition with this peptide eliminated the binding of the antibody to the lesion area (Figure 1B, right panel). Similarly the binding of the antibody was eliminated by preincubation of the primary antibody with free 3-nitrotyrosine, reduction of the tissue nitrotyrosine content with sodium dithionite, and omission of the primary antibody (not shown).
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Quantification of Protein Nitration in Aortic Lesions
Quantification of the aortic protein levels of 3-nitrotyrosine by previously established stable isotope dilution LC/ESI/MS/MS methodologies10 confirmed the changes observed by the immunohistochemical survey of the atherosclerotic lesions. The results were normalized for the precursor amino acid tyrosine and expressed as the ratio 3-nitrotyrosine/tyrosine. Protein 3-nitrotyrosine levels in LA–apoA-I–/– lesions were significantly increased as compared with the LA, 2.6±0.4 versus 0.4±0.1 mmol 3-nitrotyrosine/mol tyrosine, mean±SD (P<0.001; Figure 1C).
Localization of Tyrosine Nitrated Proteins by Immunoelectron Microscopy
The subcellular localization of protein targets in the aortic root sections were further examined by high-resolution electron microscopy using antinitrotyrosine antibodies and protein A gold labeling. In aortic root sections of LA–apoA-I–/– mice, increased labeling was found in the endothelial cells opposing the lesion (Figure 2A). Within the lesion area, fibroblasts and smooth muscle cells exhibited large intracellular and extracellular lipid deposits. The fibroblasts showed dilated endoplasmic reticulum membranes, characteristic for high proliferative activity and collagen production (supplemental Figure I). Intense labeling for nitrated proteins in these cells was localized in mitochondria and with endoplasmic reticulum membranes. In the endoplasmic reticulum the labeling was strictly confined to the cytosolic site of the endoplasmic reticulum membrane and was absent from the lumen (see high magnification inset). Also the abundant extracellular collagen fibers in the lesion area were devoid of labeling with the antinitrotyrosine antibodies. In smooth muscle cells, labeling was often associated with the cytoplasmic myofilaments (supplemental Figure I). Although most of the lipid in the lesion is extracted during the sectioning and labeling procedure, it was evident that labeling for nitrated proteins was also associated with the lipid cores in these cells. The labeling in the lipid cores reflects the modified proteins rather than nitrated lipids because the affinity-purified antibodies used do not recognize nitrated lipids or nitrated lipid adducts on proteins (R. Lightfoot and H. Ischiropoulos, unpublished data, 2006). The labeling was abolished when the primary antibody was preincubated with free 3-nitrotyrosine (Figure 2C). Collectively these data indicate that apoA-I expression is critical in modulating the extent of tyrosine nitration of proteins in atherosclerotic lesions independently of HDL-C levels.
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Proteomic Profiling of Plasma Proteins Modified by Nitration
Tyrosine-nitrated proteins in plasma were enriched by immunoprecipitation. The captured proteins were separated either on a 10% 1-dimensional gel (Figure 3A) or on a 4% to 12% gradient 2-dimensional gel. Protein-stained bands were excised and digested with trypsin for LC/ESI/MS/MS. In another set of experiments, the captured proteins were trypsin digested in solution without any additional separation followed by LC/ESI/MS/MS peptide analysis. For the evaluation of MS/MS sequence-to-spectrum assignments rigorous selection criteria were used as described in detail in the Methods section.
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Using this approach we identified 17 proteins in the enriched fraction of LA–apoA-I–/– plasma (Table 1). Immunoglobulins, serum albumin, fibrinogen, and complement proteins were the predominant proteins identified in the LA–apoA-I–/– plasma. Immunoglobulins, serum albumin, and fibrinogen were found in all LA–apoA-I–/– plasma samples analyzed irrespective of the method. Two tissue-derived proteins, liver carboxyesterase N and mannose-binding protein A, were also identified in the enriched fraction. In contrast to LA–apoA-I–/–, only 4 proteins were identified in samples from LA plasma. Hemopexin and serum albumin were common between the LA and the LA–apoA-I–/– mice and complement C5 precursor and an immunoglobulin sequence, were unique for LA plasma.
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The nitration of tyrosine residues is expected to increase the mass of peptides by 45 amu. Therefore, using the sequence-to-spectrum and manual inspection criteria the MS/MS data were evaluated for the presence of tyrosine residues with an increase by 45 amu. The data in Table 2 reveal the proteins and the specific peptide sequences that contain a tyrosine residue with a +45 amu modification. Thirteen specific peptide sequences belonging to 6 plasma proteins were identified. Three tyrosine residues, specifically Y60, Y36, and Y425, were found nitrated on serum albumin. These modified peptides were identified in 1 of the 2 replicates and only in LA–apoA-I–/– plasma. Tyrosine residues 365 and 425 in albumin have been recently reported as targets for nitration in vitro.17,18 Two sequences, which belong to IgG heavy chain-constant region and Ig kappa (light) chain, respectively, were identified in both models. Additional nitrated peptides that belong to several immunoglobulin subtypes were documented in LA–apoA-I–/– plasma (Table 2), but not in LA. One immunoglobulin sequence of the 13 peptides was unique to the LA plasma.
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Because immunoglobulins appear to be a major target for modification, we validated the proteomic approach, in part, by probing the nitrotyrosine-enriched fraction from LA–apoA-I–/– plasma with anti-mouse immunoglobulin antibodies. The analysis confirmed that IgG was present in the bound fraction (Figure 3B).
Effect of Tyrosine Nitration on Fibrin Clot Formation
Previously we reported increased levels of nitrated fibrinogen in coronary artery disease (CAD) subjects as compared with control healthy individuals.19 Because fibrinogen was identified as 1 of the proteins modified by nitration in the plasma of LA–apoA-I–/– mice, we sought to determine whether in vivo fibrinogen nitration alters the rate of fibrin clot formation. For these studies we used human plasma from CAD subjects. Fibrinogen was isolated from the plasma of 3 CAD subjects by glycine precipitation (Figure 4A). The isolated fibrinogen samples were then depleted from nitrated fibrinogen by immunoaffinity capture with antinitrotyrosine antibodies. As control the same isolated fibrinogen was passed through nonspecific IgG columns. The fibrinogen eluted from the bound fraction of the antinitrotyrosine columns stained positive for fibrinogen and 3-nitrotyrosine (Figure 4B), whereas bound fractions from the nonspecific IgG columns lacked reactivity for fibrinogen and 3-nitrotyrosine (Figure 4C). These results demonstrate that fibrinogen is nitrated in the plasma of CAD patients, with the beta chain being the preferential site of nitration,19 and that the nitrated molecules are effectively removed from the initial fibrinogen preparation with the antinitrotyrosine antibodies, but not with the nonspecific IgG. Using this approach we generated fibrinogen samples depleted from nitrated molecules (the unbound fractions of the antinitrotyrosine columns) and identical fibrinogen samples, which retained the nitrated molecules (the unbound fractions of the non specific IgG columns). The effect of fibrinogen nitration was then investigated by comparing the kinetics of fibrin clot formation in nondepleted and 3-nitrotyrosine-depleted samples, under the same experimental conditions. The fibrinogen concentration was adjusted to 0.4 mg/mL in TBS pH 7.4, and polymerization was initiated by the simultaneous addition of -thrombin and calcium chloride to a final concentration of 1 NIH U/mL and 5 mmol/L, respectively. The data in Figure 4D and 4F indicate a significant decline in the rate of fibrin clot formation and in final clot turbidity in the 3-nitrotyrosine–depleted samples as compared with the nondepleted samples of the same fibrinogen at identical concentration.
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Circulating Immunoglobulins That Recognize Tyrosine Nitrated Proteins
To explore the overall significance of protein tyrosine nitration we evaluated the presence of circulating antibodies that recognize nitrotyrosine. Similar to previous extensive documentation for the presence of circulating autoantibodies to various oxidized epitopes of lipoproteins in human and animal models of atherosclerosis,20–25 it has been recently shown that tyrosine-nitrated peptides are neoepitopes that induce production of immunoglobulins in humans and animal models.26–28,16 Using a previously developed and validated competition ELISA,16 data in Figure 5 reveal a significant increase in circulating immunoglobulins that recognize nitrotyrosine in the LA–apoA-I–/– plasma as compared with the LA plasma. The data indicate that a potential functional consequence of protein tyrosine nitration in this model of atherosclerosis includes the induction of humoral responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of apoA-I in modulating the oxidative protein nitration burden that is associated with the development of atherosclerosis was evaluated in atherosclerosis-prone mice that specifically lack apoA-I. Immunohistochemical detection revealed increased formation of nitrated proteins in LA–apoA-I–/– as compared with LA mice, implying an association between apoA-I expression and protection from nitrating oxidants. Immunoelectron microscopy revealed specific intracellular localization of nitrated proteins in endothelium, fibroblasts, and smooth muscle cells within the lesion area of these mice, including the lipid core. These observations were further confirmed by quantification of protein 3-nitrotyrosine in aortic roots using validated mass spectrometric methodology, which revealed a significant increase in protein 3-nitrotyrosine in the lesion of the LA–apoA-I–/– mice. Furthermore, using a proteomic approach we identified the protein targets for tyrosine nitration in plasma, and for several of these proteins the specific tyrosine residue(s) modified in the polypeptide chain were also identified. To investigate the potential consequences of tyrosine nitration we further documented the induction of humoral immune responses against nitrated neoepitopes and an increase in the kinetics of fibrin clot formation. Collectively, the data in these mouse models of atherosclerosis support the concept that the presence of apoA-I significantly reduces the oxidative burden and possibly contributes in part to the antiatherosclerotic effects of apoA-I.
The formation of nitrating and chlorinating oxidants capable of modifying proteins has been documented in human atherosclerosis. The levels of 3-nitrotyrosine are increased in both atherosclerotic lesions29 and plasma of coronary artery disease (CAD) patients.30 Moreover, apoA-I extracted from the lesions of CAD patients was modified by tyrosine nitration and chlorination.10,30 Subsequent in vitro studies revealed that tyrosine chlorination and methionine oxidation in apoA-I were primarily responsible for the decreased ATP binding cassette transporter 1–mediated cholesterol efflux.31–33
By using recently developed proteomic approaches we identified the proteins targeted for nitration in the plasma of the LA–apoA-I–/– mice during lesion formation. The identification of the specific targets for tyrosine nitration provides 2 potential molecular mechanisms relevant to the development of atherosclerosis in these mice. The first relates to the consequence in protein function as a result of the oxidative modifications. Previous data indicated that nitration of fibrinogen in vitro accelerated fibrin clot formation and increased levels of nitrated fibrinogen were found in subjects with coronary artery disease.19 These data performed with fibrinogen extracted from human subjects (Figure 4) that was nitrated in vivo provide direct evidence that tyrosine nitration results in accelerated fibrin clot formation. Although associations between elevated fibrinogen levels and the risk for coronary heart disease and in some cases of premature death from cardiovascular disease34–36 have been documented in epidemiological studies, a causative correlation between high levels of fibrinogen and cardiovascular disease has not been firmly established. Undesired conversion of fibrinogen to fibrin and deposition of both fibrinogen and fibrin onto atherosclerotic plaques is a well-recognized process that leads to the formation of the fibrous cap. A major and devastating complication is the disruption of the fibrous cap from the plaque that leads to often-fatal myocardial infarction or stroke.37 Therefore acceleration of fibrin clot formation and increased clot density, which reflects altered fibrin clot architecture,38 could account for the increased propensity for thrombotic events in CAD subjects.
It has been previously shown that autoantibodies recognizing oxidized epitopes in lipoproteins are present in human and animal models of atherosclerosis.20–25 Similarly we have recently reported the presence of circulating antibodies that recognize tyrosine nitrated proteins in humans,16 and it is well known that tyrosine nitrated peptides are potent inducers of antibody production. Recent data indicated that nitration of specific tyrosine residues on proteins and peptides, which are not immunogenic, can render them highly immunogenic.26,27 In addition, injection of mice with autologous nitrated IgG resulted in antibody formation that cross-react with single stranded DNA in a model of systemic lupus erythematosus.28 The presence of several peptide sequences of IgG that were modified by nitration implies that immune responses could be elicited in the LA–apoA-I–/– mice. The data in Figure 5 indicate that indeed LA–apoA-I–/– mice have increased circulating immunoglobulins that specifically recognize 3-nitrotyrosine. The humoral response to self-antigens may have an important role in normal catabolic processes that remove undesired antigens. For example, antibodies to various oxidized forms of low-density lipoprotein (LDL) may help in eliminating these potentially toxic lipids and reduce atherosclerosis.39–42 However in other circumstances the presence of autoantibodies, for example autoantibodies to IL-8 were shown to have proinflammatory activity and were associated with unfavorable outcomes.43 Therefore the biological significance of the humoral response against tyrosine-nitrated proteins in the setting of atherosclerosis requires further experimentation to ascertain whether it is beneficial or harmful for the host.
Although animal models of atherosclerosis exhibit noticeable differences from human disease the generation of genetically manipulated mice that selectively eliminate or overexpress proteins of interest provides new opportunities for investigating the specific role of these proteins in the mechanisms of the disease. Specifically the LA–apoA-I–/– mice, which exhibit only a modest decrease of HDL, are suitable for the evaluation of the antiatherogenic role of apoA-I independently of HDL. The data presented herein indicated that in the absence of apoA-I there is a considerably higher oxidative burden reflected by the modification of specific proteins. Although it is not possible to specifically ascribe the lower oxidative burden in the apoA-I competent mice to an antioxidant effect or to the previously described antiinflammatory function, the data clearly support a protective role for apoA-I. In the absence of this protection the oxidative modifications of critical proteins might disturb the fine balance between inflammatory stimuli, endothelial barrier function, and cellular viability, leading to dysfunctional endothelium, deregulation of the immune system, and progression of atherogenesis.
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Acknowledgments |
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Sources of Funding
The work was supported by NIH grants PO1 HL 076491 and P01 HL77107 (to S.L.H.), ES013125 (to D.L.), P50 HL70128 (to D.R. and H.I.), and RO1 HL54926 (to H.I.). D.M.F. is a recipient of a postdoctoral fellowship from CNPq-Brazil.
Disclosures
None.
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Footnotes |
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Original received April 17, 2007; resubmission received June 8, 2007; accepted June 25, 2007.
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