The oxidation status of HDL plays an important role in determining how this lipoprotein prevents or promotes atherosclerosis. Since patients with normal levels of HDL experience atherogenic events (ie., stroke, myocardial infarction), HDL function itself may be a stronger indicator of cardiovascular disease than the actual levels of HDL cholesterol. Bioassays of HDL function are complex, time-consuming, technician- and technique-dependent and difficult to reproduce between labs. Thus, highly efficient assays of HDL function are desperately needed. In this application, we hypothesize that the biophysics of HDL interactions with biomolecules the mediate HDL-dependent cholesterol metabolism represent a novel source of physiological information that can be used to assess the functional state of HDL. Using biolayer interferometry (BLI), a new label-free technique for measuring biomolecular interactions, we will develop a series of novel assays of HDL """"""""function"""""""" to determine if and the extent to which oxidation impacts on the ability of HDL to interact with 1) anti- and pro-inflammatory enzymes (paraoxonase [PON1], platelet activating factor acetylhydrolase [PAF-AH], myeloperoxidase [MPO] and xanthine oxidase [XO]) to prevent LDL oxidation;2) or ATP binding cassette (ABC) transporter 1 (ABCA1)/ABCG1, lecithin cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP) and scavenger receptor class B1 (SR-B1) to mediate HDL-cholesterol release, esterification, transfer and uptake, respectively. The mechanisms by which HDL is anti-inflammatory or participates in reverse cholesterol transport is directly dependent on the ability of HDL to bind these biomolecules. As such, any oxidation-induced changes in HDL binding affinity for these biomolecules should provide a sensitive index of HDL functionality.
In Aim 1, we propose to measure the extent to which oxidized lipids and proteins in reconstituted HDL (r-HDL) impair HDL function using established in vitro bioassays of cholesterol release, esterification, transfer and uptake.
In Aim 2, we will ue BLI assays to determine how oxidized lipids and proteins in r-HDL alter binding rates and affinity for the proteins and enzymes that mediate HDL's ability to inhibit LDL oxidation or promote specific steps in the reverse cholesterol transport pathway.
In Aim 3, we propose to verify and validate that BLI assays by 1) correlating BLI assays with bioassays of HDL function in established murine models of vascular disease and 2) by using BLI assays to predict which patients have clinically-diagnosed atherosclerosis in case-controlled, blinded human studies. Successful completion of these studies will lay the foundation for the development of a new clinical assay for determining HDL functionality. Validation of these new assays will allow us to identify which patients have dysfunctional HDL in a time frame and for a cost that is compatible with clinical reference laboratories. Development of these novel assays will make it possible, for the first time, to perform large clinical studies to fully test the idea that dysfunctional HDL is better indicator of atherosclerotic risk.

Public Health Relevance

Recent reports indicate that assays that measure the function of HDL, the good cholesterol, are better predictors of who will develop heart disease than measurements of HDL cholesterol. Unfortunately bioassays of HDL function are complex and time consuming, meaning that they will never be used by hospitals as diagnostic tests. In this application we propose to develop novel in vitro assays of HDL function based on the ability of HDL to bind biomolecules that are involved in HDL-dependent protection against inflammation and cholesterol metabolism. Developing this new line of in vitro assays to assess HDL functionality will not only provide critical new insight into the atherogenic status of a patient bt also answer an unmet national need in health care.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL112270-02
Application #
8511809
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Liu, Lijuan
Project Start
2012-07-16
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$628,118
Indirect Cost
$121,761
Name
Medical College of Wisconsin
Department
Surgery
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Weihrauch, Dorothee; Krolikowski, John G; Jones, Deron W et al. (2017) Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice. J Vis Exp :
Afolayan, Adeleye J; Alexander, Maxwell; Holme, Rebecca L et al. (2017) Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function. J Biol Chem 292:2369-2378
Sorci-Thomas, Mary G; Thomas, Michael J (2017) AIBP, NAXE, and Angiogenesis: What's in a Name? Circ Res 120:1690-1691
Strzepa, Anna; Pritchard, Kirkwood A; Dittel, Bonnie N (2017) Myeloperoxidase: A new player in autoimmunity. Cell Immunol 317:1-8
Hoekstra, Menno; Sorci-Thomas, Mary (2017) Rediscovering scavenger receptor type BI: surprising new roles for the HDL receptor. Curr Opin Lipidol 28:255-260
Zhang, Hao; Ray, Avijit; Miller, Nichole M et al. (2016) Inhibition of myeloperoxidase at the peak of experimental autoimmune encephalomyelitis restores blood-brain barrier integrity and ameliorates disease severity. J Neurochem 136:826-836
Yu, Guoliang; Liang, Ye; Huang, Ziming et al. (2016) Inhibition of myeloperoxidase oxidant production by N-acetyl lysyltyrosylcysteine amide reduces brain damage in a murine model of stroke. J Neuroinflammation 13:119
Pollard, Ricquita D; Fulp, Brian; Sorci-Thomas, Mary G et al. (2016) High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation. Biochemistry 55:4971-81
Yu, Guoliang; Liang, Ye; Huang, Ziming et al. (2016) Erratum to: Inhibition of myeloperoxidase oxidant production by N-acetyl lysyltyrosylcysteine amide reduces brain damage in a murine model of stroke. J Neuroinflammation 13:166
Kaul, Sushma; Xu, Hao; Zabalawi, Manal et al. (2016) Lipid-Free Apolipoprotein A-I Reduces Progression of Atherosclerosis by Mobilizing Microdomain Cholesterol and Attenuating the Number of CD131 Expressing Cells: Monitoring Cholesterol Homeostasis Using the Cellular Ester to Total Cholesterol Ratio. J Am Heart Assoc 5:

Showing the most recent 10 out of 38 publications