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
To investigate the crucial role of platelet-derived thromboxane A2 (TXA2) in initiating Ag-specific contact sensitivity (CS), a platelet-dependent CS model using genetically mast cell-deficient W/Wv mice, was provided. In vivo treatment with BAYu3405, a TXA2 receptor antagonist, markedly suppressed CS responses in a dose-dependent manner. This inhibitory effect occurred when BAYu3405 was administered before an early initiating phase, suggesting that TXA2 may be a potent initiator of platelet-mediated CS responses. When platelets were pretreated with BAYu3405 in vitro, platelet aggregation as well as serotonin release, which is able to induce the early phase response allowing local recruitment of CS effector T cells due to direct activation of vascular endothelial cells, was inhibited. The addition of U46619, a TXA2 agonist, or a mixture of platelets and thrombin-enhanced expression of both ICAM-1 and VCAM-1 on isolated mouse aortic endothelial cells, which was completely abolished by pretreatment with BAYu3405. Furthermore, intradermal injection of U46619 into the ear of platelet-depleted mice led to CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. These findings suggest that TXA2 generated from platelets activated with Ag may mediate initiation of CS responses through inducing serotonin release from platelets and the subsequent aggregation and up-regulated expression of ICAM-1 and VCAM-1 on vascular endothelial cells.
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