Our long term objectives are to understand in molecular detail cellular processes which lead to atherosclerotic disease and to use this understanding to help develop rational approaches for the diagnosis, treatment and prevention of atherosclerosis. Atherosclerotic plaques contain macrophages which serve as scavenger cells that accumulate cholesterol from plasma lipoproteins. The cholesterol-laden macrophages are calfoam cells. The goal of the proposed research is the establishment of a somatic cell system for the biochemical and genetic analysis of macrophage-specific lipoprotein metabolism and cloning of a gene for a macrophage-specific lipoprotein receptor. We have previously developed a method to transfer functional genes for lipoprotein receptors into cells that lack the receptors. The method combines DNA-mediated gene transfer with subsequent treatment of the cells with selective medium. The selective medium kills cells that do not express the lipoprotein receptors. For example, we have used this approach to introduce functional human LDL receptors into LDL receptor-deficient Chinese hamster ovary (CHO) cell mutants. The focus of this proposal is the acetyl LDL receptor, a macrophage-specific lipoprotein receptor that mediates the endocytosis of modified lDL and the formation of foam cells in culture. CHO cells do not normally express the macrophage-specific acetyl LDL receptor. In preliminary experiments, we have used DNA transfer and lipoprotein receptor selection to isolate acetyl LDL receptor bearing CHO cells.
The aim of the proposed research is to characterize in detail the lipoprotein and cholesterol metabolism of the selected cells and to compare the properties of these cells to those of true macrophages. The selected cells might be ideally suited for the somatic cell genetic analysis of macrophage-specific lipoprotein endocytosis and foam cell formation. In addition, the human acetyl LDL receptor gene will be cloned from secondary DNA-transfectants derived from these cells. Human-specific (Alu-family) repetitive DNA will be used as a probe for detecting acetyl LDL receptor genomic clones in a phage library. These genomic clones will in turn be used to isolate cDNA which will be sequenced to determine the primary structure of the acetyl LDL receptor. The methods developed to isolate CHO cells that express the acetyl LDL receptor. The methods developed to isolate CHO cells that express the acetyl LDL receptor should prove to be useful for the characterization of other tissue-specific receptors which mediate endocytosis.
