A novel assay to measure low-density lipoproteins binding to proteoglycans

• Published in: PloS one Vol. 19; no. 1; p. e0291632
• Main Authors: Geh, Esmond N, Swertfeger, Debi K, Sexmith, Hannah, Heink, Anna, Tarapore, Pheruza, Melchior, John T, Davidson, W Sean, Shah, Amy Sanghavi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.01.2024; Public Library of Science (PLoS)
• Subjects: Adults; Affinity; Analysis; Analysis and chemistry; Animals; Antibodies; Apolipoproteins; Apolipoproteins B - metabolism; Arteriosclerosis; Assaying; Atherosclerosis; BASIC BIOLOGICAL SCIENCES; Binding; Binding analysis; Biology and Life Sciences; Blood; Blood lipids; Cardiovascular disease; Cardiovascular Diseases; Care and treatment; Cell binding; Cell binding assay; Cell culture; Cholesterol, LDL; Chondroitin; Chondroitin sulfate; Coronary vessels; Density; Diabetes; Diagnosis; Enzyme-linked immunosorbent assay; Ethylenediaminetetraacetic acid; Extracellular matrix; Health aspects; Heparan sulfate; Immunofluorescence; Laboratories; Lipoproteins; Lipoproteins, LDL - metabolism; Low density lipoprotein; Low density lipoproteins; Measurement; Medicine and Health Sciences; Methylene blue; Mice; Physical Sciences; Protein Binding; Proteins; Proteoglycans; Proteoglycans - metabolism; Research and Analysis Methods; Risk factors; Smooth muscle; Sulfates; Type 2 diabetes; United States; Indiana; United States > US; Fiji
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0291632

The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.