Lipoprotein Uptake and Metabolism by Rat Aortic Smooth Muscle Cells in Tissue Culture

Abstract

Aortic smooth muscle cells from the rat were successfully grown in tissue culture and shown to have characteristic morphology. 125 I-labeled homologous very low density lipoproteins and high density lipoproteins were taken up by these smooth muscle cells during incubation for 48 hours at the stationary phase. Despite multiple washings, a large proportion of the lipoprotein radioactivity associated with the cells was apparently surface bound and trypsin releasable. With both lipoprotein fractions, lipid and protein uptake by the cells measured after trypsinization was related to time and to the amount of lipoprotein protein added to the medium. Compared with protein, there was a disproportionately greater entry of lipid radioactivity into the cells. Light and electron microscope autoradiography localized the label intracellularly over the cell cytoplasm, cell boundaries, and, in some cells, over lysosomes. On the basis of either protein uptake or whole particle uptake, approximately four times as much high density lipoprotein as very low density lipoprotein was taken up by the smooth muscle cells. To assess metabolism and degradation of high density lipoproteins, aortic smooth muscle cells were incubated in fresh unlabeled medium for 48 hours after exposure to 125 I-labeled high density lipoproteins. A large proportion of radioactivity released was trichloroacetic acid precipitable, suggesting some release of whole lipoprotein protein; however, these lipoproteins appeared to be modified when they were tested with anti-high density lipoprotein antiserum. Also, water-soluble radioactivity (presumably protein breakdown products) was released in amounts that averaged 3% of the protein label in the cells. These results indicate that although aortic smooth muscle cells growing in tissue culture can rapidly take up lipids and lipoproteins, catabolism of lipoprotein protein is slow. Correlative biochemical and ultrastructural analysis suggests the possibility of regurgitation of noncatabolized lipoprotein protein by reverse endocytosis.