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In this study a two-compartment culture model of arterial intima was used for the in vitro induction of fatty streaklike lesions. The apparatus consisted of upper and lower compartments separated by a human amnion membrane stretched between them. Human umbilical vein endothelial cells (HUVECs) were cultured to confluence on the stromal surface of the amnion membrane. Maximal migration of blood mononuclear cells (MCs) through the HUVEC monolayer in response to a f-Met-Leu-Phe gradient was observed at 10(-8) mol/l; the migration was 3.29 times greater than that observed under the condition of random migration (control). In the study of MC transformation into lipid-laden cells in the amnion membrane (foam cell formation in 'arterial intima'), 10(6) MCs were incubated, in the presence of freshly prepared low-density lipoprotein (LDL; 100 microgram/ml). The lipid loading of MCs was time dependent. After 12 hours' incubation, 39% of the MCs that migrated into the amnion membrane contained a small number of lipid droplets, whereas the remaining 61% showed no lipid droplets. Only 1.7% of the cells contained a high number of lipid droplets in the cytoplasm and took on the appearance of foam cells. With time, the number of lipid-laden cells and the amounts of intracytoplasmic lipid droplets gradually increased. At 72 hours after incubation, 65.4% of the MCs were loaded with lipid droplets, and 20.9% of them, an eightfold increase over 12 hours of incubation, showed a foamy cell appearance. Because MCs consist of 70% monocytes and 30% lymphocytes, about 93% of the monocytes were filled with lipid after a 72-hour incubation. Ultrastructural examination showed that lipid-laden cells took on macrophage characteristics, such as wide and heterogeneous cytoplasm, indented nuclei, and abundant lysosomes. A minority of the MCs in the amnion were considered lymphocytes; they had scanty cytoplasm, round nuclei with abundant heterochromatin, no lysosomes, and no lipid vacuoles. In conclusion, the formation of an in vitro fatty streaklike lesion is demonstrated, and this is reminiscent of in vivo human atherogenesis.

Endothelial cells were cultured from human aortas and inferior venae cavae of autopsied subjects ranging in age from infancy to 85 years. Endothelial cells in 32 of more than 100 attempted cultures were pure enough for evaluation. Emerged endothelial cells in primary culture were classified into two types: typical endothelium and variant endothelium. Typical endothelial cells were small, round to polygonal shaped, and were arranged uniformly. Their diameter ranged from 50 to 70 microns. Variant endothelial cells were larger, ranging from 100 to 200 microns in diameter, and giant endothelial cells measuring more than 250 microns in diameter were scattered among them. Variant endothelial cells were usually multinucleated and possessed endothelium-specific markers of vWF and Weibel-Palade bodies. No incorporation of [3H]thymidine was found in the nuclei of cultured variant endothelial cells. Although most cultured endothelial cells were of the typical type, variant endothelial cells were interspersed throughout the culture. The ratio of variant endothelial cells to typical cells correlated well with the severity of atherosclerosis, but less so with aging. The number of variant endothelial cells in cultures from inferior venae cavae was slight and constant throughout all age groups. The presence of multinucleated endothelial cells in in vivo aortas was confirmed by both scanning and transmission electron microscopy. They sometimes existed in colonies in the aortas from elderly subjects with intimal-thickened or advanced atherosclerotic lesions. These results indicate that variant endothelial cells were present in vivo and their ratio in primary culture reflected the in vivo population. It is likely that these cells were formed by adhesion of adjacent typical endothelial cells and that this process was affected more by atherosclerosis than by aging. Although it is not clear if the multinucleated variant cells were formed before the formation of atherosclerotic plaque or after the plaque formation, they will contribute to further development of atherosclerotic lesions, which in turn cause malfunction of the cell membrane. We suggest that there is a cyclic effect of these processes for multiplication of the variant endothelial cells and advancement of atherosclerotic lesions.