Systematic RNA-interference in primary human monocyte-derived macrophages: A high-throughput platform to study foam...

Area of Science:

• Cardiovascular Biology
• Cell Biology
• Genetics

Background:

• Macrophage-derived foam cells are central to atherogenesis and coronary artery disease (CAD).
• Understanding macrophage lipid uptake is critical for identifying new therapeutic targets for CAD.
• Existing knowledge of genes regulating lipid accumulation in macrophages is incomplete.

Purpose of the Study:

• To develop and apply a high-throughput screening workflow to identify genes influencing low-density lipoprotein (LDL) uptake in primary human macrophages.
• To investigate the impact of gene silencing on LDL uptake in macrophages from patients with CAD and healthy controls.
• To explore novel molecular mechanisms, including signaling pathways, involved in macrophage lipid accumulation.

Main Methods:

• Established a 384-well plate-based workflow for siRNA transfection, macrophage differentiation, and quantitative LDL uptake measurement.
• Utilized automated microscopy and image analysis for high-throughput assessment of LDL uptake.
• Screened the impact of silencing 89 genes on LDL uptake in primary macrophages from CAD patients and controls.

Main Results:

• Identified four novel genes (APOC1, CMTM6, FABP4, WBP5) whose silencing significantly reduced macrophage LDL uptake.
• Demonstrated that knockdown of chemokine receptor CXCR4 reduced LDL uptake.
• Found that the MIF/CXCR4 signaling pathway, independent of CXCL12, plays a role in regulating macrophage lipid uptake.

Conclusions:

• The study introduces a robust high-throughput strategy for functional gene screening in primary human cells from CAD patients.
• Novel candidate genes (APOC1, CMTM6, FABP4, WBP5) impacting macrophage lipid uptake were identified.
• The macrophage-induced factor (MIF)/CXCR4 signaling pathway is implicated in regulating lipid uptake in macrophages, offering potential therapeutic avenues for CAD.