An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation

Abstract

Abstract-To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of foam cells in vitro, we have developed a 2-coculture system with rabbit aortic endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixture of matrix proteins on polyethylene filters in chemotaxis chambers. AECs were seeded on a mixture of type I and IV collagen with or without various types of serum lipoproteins (method 1) or on matrix proteins secreted by SMCs (method 2). In these coculture systems, rabbit AECs can maintain a well-preserved monolayer for up to 2 weeks. When human CD14-positive monocytes were added to the upper medium of the system, with monocyte chemotactic protein-1 treatment Ϸ60% of the monocytes transmigrated within 24 hours and were retained for up to 7 days, whereas without MCP-1 treatment, Ͻ30% of monocytes transmigrated. On day 1, transmigrant monocytes were negative for immunostaining of type I and II macrophage scavenger receptors but by day 3, became positive for scavenger receptors as well as other macrophage markers. When oxidized low density lipoprotein was added to the matrix layer of the method I coculture, on day 4 transmigrant cells exhibited lipid deposit droplets, and by day 7, they had the appearance of typical foam cells. Some of the transmigrant cells recovered in the lower medium on day 7 also appeared to be foam cells, indicating foam cell motility and escape from the coculture layer through the filter. In summary, this coculture system is a useful in vitro tool to dissect the cellular and molecular events that make up the process of foam cell formation. (Arteriosclerosis and

A Comparison of Differences in the Gene Expression Profiles of Phorbol 12-myristate 13-acetate Differentiated THP-1 Cells and Human Monocyte-derived Macrophage

“…Also, it has been reported that in certain subtypes, THP-1 cells do not express scaven-ger receptor activity upon PMA treatment (7). Furthermore, the induction of the liver x receptor alpha (LXRα) gene is not observed in THP1PMA cells, in contrast to our previous reported finding of the induction of this important receptor gene in macrophages (8). We studied the gene expression profile of THP1PMA cells and compared it with the expression profile of macrophages by means of oligonucleotide microarray analysis.…”

“…To differentiate THP-1 cells, PMA (SIGMA) was added to a final concentration of 0.2 µm and cultured for 72 hours or 7 days. Peripheral blood monocyte cells (PBMC) were isolated from normal human healthy volunteers and enriched by centrifugation with Ficoll-Paque Plus (Pharmacia Biotech, Uppsala, Sweden), and then were separated using magnetic beads conjugated with anti-human CD14 antibody as described previously (8). Some of them were immediately used for microarray analysis.…”

Genomic Structure and Mapping of Human Orphan Receptor LXR Alpha : Upregulation of LXRa mRNA During Monocyte to Macrophage Differentiation

“…Oxidative modification of low density lipoprotein and successive uptake by macrophages leads to foam cell formation, which is a key cellular event during atherogenesis (5). During atherogenesis, monocytes transmigrate into vessel walls where they differentiate into macrophages and take up modified lipoproteins (6). During the course of the analysis of gene expression profile of monocyte/macrophage, we found that LXRa is profoundly induced in macrophages differentiated in the presence of GM-CSF.…”

Deficiency of cathepsin S reduces atherosclerosis in LDL receptor–deficient mice

“…Rabbit aortic SMCs and ECs were cocultured as described (28). Briefly, 1 × 10 6 rabbit aortic SMCs were cultivated with EGM-2 media (containing 2% rabbit plasma) on the filter for more than 3 weeks.…”