Examining Heart Disease and Cholesterol with Fluorescence-Based Cholesterol Efflux Assays

Various studies have found out a negative correlation between cardiovascular disease and cellular cholesterol efflux. Reverse cholesterol transport (RCT) is a process by which the cholesterol efflux from peripheral cells and tissues occurs. RCT permits the extrahepatic cells, including macrophage-derived foam cells in arterial atherosclerotic plaque, to transfer excessive cholesterol back to the liver for bile acid synthesis and excretion, thereby decreasing the peripheral lipid burden.

Conventional protocols for quantitation of cholesterol efflux include labeling of cells with tritiated cholesterol and measuring the release of labeled sterol in a 12- or 24-well plate. However, this protocol is not suited for high-throughput screening of huge numbers of small molecules/screening compounds or serum samples for their impact on cholesterol efflux.

In this article, BioVision reports a convenient, simple, and high-throughput cell-based assay using fluorescently labeled cholesterol in a 96-well plate format. The results show that the percentage of cholesterol efflux elicited by 2% ApoB-depleted normal human serum in J774 macrophage cells is analogous to those reported in the literature. BioVision’s assay technique involves the use of a positive control for this assay, inducing efflux independent of any receptor, which is used as a measure of the reliability of the assay. This assay is greatly reproducible with low inter-assay variability (≤10%). It has been shown that this is an effective technique for measuring cholesterol efflux in a cell-based assay. The increased sensitivity of the assay along with its high-throughput screening capability allows researchers to screen large libraries of serum samples and reagents and offers a useful tool for drug discovery in the field of cardiovascular disease research.

Key Features

Examining Heart Disease and Cholesterol with Fluorescence-Based Cholesterol Efflux Assays

  • Rapid and simple protocol
  • Convenient: non-radioactive, eliminated the need for special handling or disposal
  • High-throughput
  • Highly stable: long shelf life
  • Accurate: reproducible results with low inter- and intra-assay variability
  • Fluorometric quantitation (Ex/Em = 482/515 nm)
  • Ample reagents to execute 100 assays in 96-well plate format

An In-Vitro Assay to Measure Cholesterol Efflux

(A) Cholesterol efflux, a part of reverse cholesterol transport, is a pathway transferring intracellular cholesterol from macrophage to extracellular receptors (e.g. HDL or Apo-AI). It consists of five independent routes, including ATP-binding membrane cassette transporter A1 (ABCA1), Scavenger Receptor type B1 (SR-B1) receptor, Caveolin, Cyp27A1 (Sterol 27-hydroxylase), and Passive Diffusion. (B) Cholesterol efflux was tested in macrophage cells (J774.1 cells) using BioVision’s kit. Cells were labeled with labeling media containing fluorescently labeled cholesterol for 16 hours. Cells are treated with various cholesterol acceptors like human serum, HDL (50 µg), or positive control known to cause cholesterol efflux. Cholesterol efflux is expressed as % efflux elicited by cells in 4 hours.

Figure 1. (A) Cholesterol efflux, a part of reverse cholesterol transport, is a pathway transferring intracellular cholesterol from macrophage to extracellular receptors (e.g. HDL or Apo-AI). It consists of five independent routes, including ATP-binding membrane cassette transporter A1 (ABCA1), Scavenger Receptor type B1 (SR-B1) receptor, Caveolin, Cyp27A1 (Sterol 27-hydroxylase), and Passive Diffusion. (B) Cholesterol efflux was tested in macrophage cells (J774.1 cells) using BioVision’s kit. Cells were labeled with labeling media containing fluorescently labeled cholesterol for 16 hours. Cells are treated with various cholesterol acceptors like human serum, HDL (50 µg), or positive control known to cause cholesterol efflux. Cholesterol efflux is expressed as % efflux elicited by cells in 4 hours.

Multiple Applications of In-Vitro Cholesterol Efflux Assay

  • Screen lipoprotein (recombinant or isolated) or serum samples for cholesterol efflux
  • Screen small molecules for their impact on cholesterol efflux (a useful tool for drug discovery program)
  • Measure the cholesterol efflux using different primary cells or cell lines to know the function of different acceptors or receptor molecules in cholesterol efflux (Figure 3)
  • Screen receptor-specific inhibitor(s) for cholesterol efflux (Figure 3 B)

Cholesterol efflux in Adipocytes and Endothelial Cells. (A) Cholesterol efflux was tested in adipocytes. 3T3-L1 cells (Pre-adipocytes) were differentiated in 96-well plate for 7 days using differentiation media (lipid droplets should be visible using bright field microscopy). Cells were gently washed and labeled with fluorescently labeled cholesterol. Cholesterol efflux was determined using normal serum (human) or the positive control. Fluorescence signal from non-treated cells after labeling was subtracted as background. Cholesterol efflux is expressed as % cholesterol efflux elicited by cells in 4hrs. (B) Endothelial cells (EA.hy 926 human endothelial cell line) were labeled with labeling media and treated with normal serum (human), normal serum (human) in presence of SR-BI inhibitor BLT-1 (Block Lipid Transporter -1, 5 µM), or positive control known to cause cholesterol efflux. Cholesterol efflux is expressed as % efflux elicited by cells in 4 hours. Values are mean ± SEM,

Figure 2. Cholesterol efflux in Adipocytes and Endothelial Cells. (A) Cholesterol efflux was tested in adipocytes. 3T3-L1 cells (Pre-adipocytes) were differentiated in 96-well plate for 7 days using differentiation media (lipid droplets should be visible using bright field microscopy). Cells were gently washed and labeled with fluorescently labeled cholesterol. Cholesterol efflux was determined using normal serum (human) or the positive control. Fluorescence signal from non-treated cells after labeling was subtracted as background. Cholesterol efflux is expressed as % cholesterol efflux elicited by cells in 4hrs. (B) Endothelial cells (EA.hy 926 human endothelial cell line) were labeled with labeling media and treated with normal serum (human), normal serum (human) in presence of SR-BI inhibitor BLT-1 (Block Lipid Transporter -1, 5 µM), or positive control known to cause cholesterol efflux. Cholesterol efflux is expressed as % efflux elicited by cells in 4 hours. Values are mean ± SEM, n=3. *P < 0.05 vs normal serum.

Cholesterol Efflux from Patients with Coronary Artery Diseases

(A) Cholesterol efflux was evaluated using BioVision’s Cholesterol Efflux Assay Kit in macrophage cells. J774.1 Macrophage cells were labeled with labeling media containing fluorescently labeled cholesterol and then treated with either normal human serum or with different patients serum (ApoB-depleted serum), known to have coronary artery diseases (CAD). BioVision’s assay demonstrates a significant decrease in cholesterol efflux from patients suffering from coronary artery disease compared to normal serum. Similar trend has been shown in literature where cholesterol efflux capacity has been found to be inversely associated with prevalent atherosclerotic vascular disease in four independent cohorts (2 angiographic CAD, 1 carotid intima-media thickness, and 1 outpatient clinical CAD).

Figure 3. (A) Cholesterol efflux was evaluated using BioVision’s Cholesterol Efflux Assay Kit in macrophage cells. J774.1 Macrophage cells were labeled with labeling media containing fluorescently labeled cholesterol and then treated with either normal human serum or with different patients serum (ApoB-depleted serum), known to have coronary artery diseases (CAD). BioVision’s assay demonstrates a significant decrease in cholesterol efflux from patients suffering from coronary artery disease compared to normal serum. Similar trend has been shown in literature where cholesterol efflux capacity has been found to be inversely associated with prevalent atherosclerotic vascular disease in four independent cohorts (2 angiographic CAD, 1 carotid intima-media thickness, and 1 outpatient clinical CAD).

Baseline characteristics of coronary artery patients and healthy-volunteer control

Patient ID Age Sex Cholesterol
(mg/dl)
HDL LDL/VLDL
P1     129.31 4.56 124.74
P2     88.70 1.43 87.26
P3     92.48 1.61 90.86
P4     229.38 6.07 223.30
P5     159.56 13.23 146.33
Normal Serum     140.0 40.2 100.5

 

Conclusion

  • BioVision’s Cholesterol Efflux Assay Kit is a high-throughput screening assay to measure cholesterol efflux in cells using fluorescently labeled cholesterol.
  • Various factors altering reverse cholesterol transport and cholesterol efflux can be tested with the help of this kit.
  • Cholesterol Efflux Assay offers a sensitive, safe, and reproducible technique for measuring cholesterol efflux.
  • BioVision’s Cholesterol Efflux Assay will address the significance of reverse cholesterol transport and cholesterol efflux in atherosclerosis.

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Feb 21, 2020 at 9:30 AM

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