Designing and development of a novel efflux assay based on liposome and macrophage to evaluate hdl's reverse cholesterol transport capacity

Cardiovascular disease remains a major cause of mortality and morbidity worldwide. Low high-density lipoprotein-cholesterol(HDL-C) is a significant cardiovascular risk factor. Evaluation of HDL's functional properties, mainly cholesterol efflux capacity (CEC), could be an improved measure of its cardio-protective properties. Apolipoprotein A1 binding protein (AIBP) is a secreted protein that promotes the binding of apolipoprotein A-I (ApoA-I) to ATP-binding cassette transporter A1 (ABCA1),a cell membrane protein. AIBP-ApoA1 and ABCA1 interaction increases the efflux of cholesterol by enabling the transfer of intracellular cholesterol and phospholipids to ApoA1.This interaction suggests a role in HDL functionality and inflammatory status in coronary artery disease(CAD). However,translating CEC assay into a standardized clinical assay is limited by technical variability and poor reproducibility.

We previously demonstrated a comparison between an immobilized liposome-bound gel beads (ILGs) based CEC activity assay with macrophage-based CEC activity in Acute Coronary Syndrome(ACS) patients admitted to our Institute in India. Translational research in our project aimed to develop, validate, and implement an improved HDL functionality assay or metric that can be used in routine clinical laboratories for assessing cardiovascular risk. This study also explores genetic heterogeneity in APOA1 & AIBP affecting CEC and inflammatory profiles in CAD & ACS patients.

Environmental scanning electron microscopy (ESEM), transmission electron microscopy(TEM) & confocal microscopy were done to characterise ILGs to confirm the morphology, internal porosity, & liposome encapsulation. A total of sixty CAD (25), ACS patients (25) and healthy controls (10) have been enrolled from the Cardiology emergency, after taking informed consent. Blood samples were collected for lipid profiling & inflammatory cytokine measurement. Targeted gene sequencing of APOA1 and AIBP will be done to identify sequence variation.

The characterization of beads by TEM, ESEM, & confocal microscopy revealed the porous structure of the beads & successful liposome encapsulation. ILGs were observed to be feasible for use as a cell mimetic for standardized CEC assessment. The lipid profile of patients indicates a trend toward low HDL, high LDL, high TC & moderate triglyceride elevation.We expect that since AIBP may contribute to inhibit inflammation in CAD/ACS patients, genetic heterogeneity in APOA1 and AIBP could provide a useful metric for HDL functionality in combination with liposome assay.

We suggest that ILGs have the potential to be effective cell mimetics for a liposome-based CEC assay to assess HDL functionality.This CEC assay using ILGs is more reproducible than the cell-based conventional CEC assay. This could be of value for precision atherosclerotic risk assessment.

ILGs Analysis 1