(77e) Comparing Monocyte Adhesion and Transendothelial Migration Between a 3D Vascular Tissue Model and a 2D Cell Culture System Under Normal Conditions and Activated with TNF-&alpha

Atherosclerosis is known as an inflammatory disease which is mainly initiated by accumulation of lipid substances in the subendothelial layer of major arteries, followed by adhesion and transmigration of monocytes.  This adhesion and transmigration involves several steps such as slow rolling, adhesion strengthening, crawling, paracellular and transcellular migration, and transmigration through the basement membrane, which are mediated by many bioactive molecules. The bioactive molecules include cellular adhesion molecules (CAMs) and chemotactic cytokines (chemokines). CAMs are proteins expressed on the surface of the cells and are involved in the adhesion of monocytes to the endothelial cell layer. Monocyte Chemoattractant Protein-1 (MCP-1) is one chemokine that plays an important role in monocyte trafficking across the endothelial layer. Studies have shown that chemokine concentration gradients drive and possibly control cell migration. Many studies investigate the effect of chemokines, such as MCP-1 on monocyte migration by using a 2D experimental system.  Endothelial cells are grown on a thin, microporous membrane, the chemokine is added to the aqueous media below the membrane, monocytes are added to the apical surface of the endothelial cells, and transmigration of the cells across the endothelial cell layer and the microporous membrane is observed.  The 2D system may not adequately predict in vivo cell behavior due to the lack of the third dimension. A 3D tissue model consisting of a collagen matrix would be a better experimental model to mimic the subendothelial extracellular matrix (ECM). The 3D tissue model provides the added dimension that is important for the creation of a diffusive concentration gradient formed in the ECM, which is responsible for the control of many cellular mechanisms.  The concentration gradient formed in the 3D model is distinctly different compared to the one in the 2D cell culture system, where the secreted factors from the endothelial cells dissolve quickly into the surrounding media. Apart from this advantage, when focusing on the transmigration of monocytes, this matrix can also provide an area for monocytes to localize and differentiate into macrophages after the transendothelial migration. We hypothesized that the chemokine concentration gradient within the ECM of the 3D tissue model will drive a different cellular response than that in the 2D system. The goal of this research was to measure the formation of MCP-1 concentration gradients within a 3D vascular tissue model and determine the effect on monocyte migration. The first objective was to characterize the effect of key cellular adhesion molecules on the surface of endothelial cells, known to be important for monocyte adhesion and transmigration. This was necessary in order to determine if the endothelial cell surface characteristics (kinetics, level of expression) are similar between 3D and 2D models. The experimental results for this part show that the CAMs expression of Human Aortic Endothelial Cells (HAECs) after stimulation with tumor necrosis factor-α to mimic inflammatory conditions is significantly different between the two systems. The next objective was to compare the monocyte adhesion and transmigration through the endothelial layer between the two models. Experimental results show that the expression of MCP-1 on HAECs in the 2D system was significantly higher than in the 3D system after stimulation. However, monocyte transmigration was significantly higher in the 3D system compared with the 2D system, suggesting that other mechanisms controlling cell migration may be at play. The final results of this research will provide new information about the relationship between MCP-1 concentration gradients and monocyte transendothelial migration, and will lead to the development of an improved model to study transendothelial monocyte migration associated with inflammation.