Reverse cholesterol transport: current assay methods, alterations with disease and response to therapeutic intervention

Abstract

The removal of excess cholesterol from the body by High-density lipoprotein (HDL) in a process termed reverse cholesterol transport (RCT) has long been proposed to play a critical role in reduction of the lipid burden in arterial wall atherosclerotic lesions. While HDL-cholesterol levels are associated with decreased cardiovascular risk and considered to be "good-cholesterol", clinical studies using HDL-raising therapies to potentially enhance RCT have consistently produced disappointing results. In this mini review we evaluate the effects of human disease on RCT along with the changes in this process upon various therapeutic interventions. Despite the importance of assay standardization, the major method for monitoring RCT has relied upon the cholesterol efflux capacity (CEC) assay, a highly-difficult and tedious cell culture assay, which is low-throughput and only suitable for research studies. Hence, we also briefly review several new methods to measure RCT both in vitro and in vivo, along with new cell-free alternative RCT assays, which have the potential to be developed into routine automated diagnostic assay. The benefits of HDL may yet be revealed by the use of these new high-throughput RCT assays perhaps as a screening tool for novel RCT boosting agents or as new biomarkers for cardiovascular disease risk.

Keywords: ABCA1; ApoA-1; cholesterol; inflammation; reverse cholesterol transport (RCT).

Figure 1

Cholesterol Efflux based in vitro RCT surrogate assays (A–C) and ApoA-I exchange-mediated in vitro RCT surrogate assays (D–F). Schematized representations of surrogate assays for the initial step(s) in RCT. (A) Cellular Cholesterol Efflux Capacity, the “gold standard” in vitro method to measure RCT requires days of cell culture, radiocholesterol, apoB- depleted serum and is not amenable to translation to a clinical diagnostic assay. Cell-free cholesterol efflux assays. (B) The “Non-Cellular” Cholesterol Efflux Capacity assay replaces cells with immobilized liposome-bound gel beads (ILG) as a donor of fluorescent cholesterol to apoB- depleted plasma and requires overnight incubation. (C) The Cholesterol Uptake Capacity is an automated multi-step assay that requires apoA-I-mediated capture of Biotin-PEG3-cholesterol- labeled serum HDL onto magnetic beads and then detection of luminescence of biotin-bound alkaline phosphatase-conjugated streptavidin. (D) Exchange of exogenous apoA-I to plasma HDL: The HDL-apoA-I Exchange assay measures electron paramagnetic resonance of exogenous nitroxide spin-labeled apoA-I that exchanges onto plasma HDL. (E) The ApoA1 Exchange Rate assay measures the kinetics of dual fluorescent emission of plasma HDL- associated exogenous apoA-I labeled with NBD (cholesterol-sensitive) and Alexa647 (non- cholesterol-sensitive reference). (F) The HDL-specific phospholipid efflux assay monitors solubilization by endogenous apoA-I and other exchangeable HDL apolipoproteins in whole plasma, of a non-transferable fluorescent phospholipid from phospholipid/cholesterol coated nanoparticles. Figure created in BioRender. Zubiran, R. (2025) https://BioRender.com/k10f072.