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 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 approximately 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 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.
Takaku
M
M
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
Wada
Y
Y
Jinnouchi
K
K
Takeya
M
M
Takahashi
K
K
Usuda
H
H
Naito
M
M
Kurihara
H
H
Yazaki
Y
Y
Kumazawa
Y
Y
Okimoto
Y
Y
Umetani
M
M
Noguchi
N
N
Niki
E
E
Hamakubo
T
T
Kodama
T
T
D000818:Animals; D001011:Aorta / Q000166:cytology; D001161:Arteriosclerosis / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000276:immunology*; D018932:Chemokine CCL2 / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D005487:Foam Cells / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008297:Male; D008855:Microscopy, Electron, Scanning; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D011817:Rabbits; D012835:Silver Nitrate; D016622:Silver Staining
D018932:Chemokine CCL2; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D003094:Collagen; D012835:Silver Nitrate