Differential Association of Hemoglobin with Proinflammatory High Density Lipoproteins in Atherogenic/Hyperlipidemic Mice: A NOVEL BIOMARKER OF ATHEROSCLEROSIS

Alan M. Fogelman,Junji Watanabe,Srinivasa T. Reddy,Victor Grijalva,Kathy Kozak,Hsiang-Hui Meng,Susan Hama,Helen Ge,Mohamad Navab,James C. Liao,Robin Farias-Eisner,Katherine J. Chou,Georgette Buga,Julian P. Whitelegge,Yunan Miao,Terry D. Lee

doi:10.1074/jbc.m702163200

Alan M. Fogelman, Junji Watanabe + Show 14 more

Open Access

https://doi.org/10.1074/jbc.m702163200

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Abstract

Studies in both mice and humans suggest that the anti- or proinflammatory nature of high density lipoprotein (HDL) may be a more sensitive predictor of risk for coronary heart disease events. In this study, we report the identification and characterization of two proteins (m/z 14,900 and 15,600) that are most dramatically associated with HDL in mouse models of atherosclerosis. Mass spectral analyses of proinflammatory HDL identified the two peaks to be hemoglobin (Hb) alpha and beta chains, respectively, with no apparent post-translational modification. Biochemical analysis confirmed the differential association of Hb with HDL from hyperlipidemic mice. We further show that HDL-associated Hb is predominantly in the oxyHb form with distinct physical and chemical properties. Furthermore oxyHb-containing proinflammatory HDL potently consumed nitric oxide and contracted arterial vessels ex vivo. Moreover Hb also was found differentially associated with HDL from coronary heart disease patients compared with healthy controls. Our data suggest that Hb contributes to the proinflammatory nature of HDL in mouse and human models of atherosclerosis and may serve as a novel biomarker for atherosclerosis.

Highlights

Atherosclerosis is the leading cause of morbidity and mortality in Western society
We demonstrate that the association of Hb with high density lipoprotein (HDL) plays an important role in the modulation of time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid chromatography; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography; PBS, phospate-buffered saline; Isoelectric focusing (IEF), isoelectric focusing; HRP, horseradish peroxidase; ACh, acetylcholine; MALDI, matrix-assisted laser desorption ionization; Hp, haptoglobin; Hx, hemopexin; proliNONOate, 1-(hydroxy-NNO-azoxy)-L-proline
Individual HDL samples obtained from D7 and W15 groups on chow or atherogenic diet were subjected to anion exchange (Q10) fractionation

Summary

Animal Experiments

Wild-type, low density lipoprotein receptor-deficient, and apoE null C57BL6/J wild-type female mice at an age of 8 –12 weeks were used in experiments comparing the three mouse models. Mice were fed one of three diets for the periods described: chow diet (Ralston Purina Mouse Chow); atherogenic diet containing 15.8% fat, 1.25% cholesterol, and 0.5% cholate (Harlan Teklad, Madison, WI); or a western diet containing 42% fat and 0.15% cholesterol (Harlan Teklad). For short term studies mice were fed the described diet for 7 days (D7), and for long term studies mice were fed the described diet for 15 weeks (W15). Serum samples were isolated from overnight fasted mice, cryopreserved in 10% sucrose, and freshly frozen at Ϫ80 °C until use

Lipoprotein Isolation

Assays to Determine the Inflammatory Properties of HDL

SELDI ProteinChip Analysis

Electrophoresis and Immunoblots

Vasoreactivity Studies

RESULTS

Hb mass values from MALDI analysis of proinflammatory HDL

HDL RBCs

DISCUSSION