An in vitro coculture model of transmigrant monocytes and foam cell formation

To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of fo am cells in vitro, we have developed a 2-coculture system with rabbit aorti c endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixtur e of matrix proteins on polyethylene filters in chemotaxis chambers. AECs w ere seeded on a mixture of type I and IV collagen with or without various t ypes of serum lipoproteins (method 1) or on matrix proteins secreted by SMC s (method 2). In these coculture systems, rabbit AECs can maintain a well-p reserved monolayer for up to 2 weeks. When human CD14-positive monocytes we re added to the upper medium of the system, with monocyte chemotactic prote in-1 treatment approximate to 60% of the monocytes transmigrated within 24 hours and were retained for up to 7 days, whereas without MCP-1 treatment, <30% of monocytes transmigrated. On day 1, transmigrant monocytes were nega tive for immunostaining of type I and II macrophage scavenger receptors but by day 3, became positive for scavenger receptors as well as other macroph age markers. When oxidized low density lipoprotein was added to the matrix layer of the method I coculture, on day 4 transmigrant cells exhibited lipi d deposit droplets, and by day 7, they had the appearance of typical foam c ells. 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 f rom the coculture layer through the filter. In summary, this coculture syst em is a useful in vitro tool to dissect the cellular and molecular events t hat make up the process of foam cell formation.